Plants and plant cells expressing histidine tagged intimin

ABSTRACT

This invention satisfies needs in the art by providing intimin, the Enterohemorrhagic  Escherichia coli  (EHEC) adherence protein, alone or as a fusion protein with one or more other antigens, expressed by transgenic plants and the use of those plants as vehicles for stimulating a protective immune response against EHEC and the one or more other antigens. Various plant species are transformed to protect various animal species and also humans against EHEC, against pathogens expressing intimin-like proteins, and against pathogens expressing any of the one or more other antigens to which intimin may be fused. 
     The eae gene encoding intimin, a functional portion thereof, or a recombination that encodes a fusion protein is put under the control of a constitutive plant promoter in a plasmid and the plasmid is introduced into plants by the type of transformation appropriate for the particular plant species. The engineered plants expressing intimin or the intimin fusion protein are then fed to animals and/or humans to elicit the production of antibodies, which protect the animals/humans against EHEC colonization and infection, and against pathogens expressing the one or more other antigens and any cross-reactive antigens. The invention may also be practiced by expressing the intimin or intimin fusion protein in other host organisms such as bacteria, yeast, and fungi.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/840,466, filed Apr. 18,1997 now U.S. Pat. No. 6,261,561, which is a continuation of provisionalapplication Ser. No. 60/015,657, filed Apr. 19, 1996 and provisionalapplication Ser. No. 60/015,938, filed Apr. 22, 1996, all of which arespecifically incorporated herein by reference.

This application is related to provisional applications entitledHISTIDINE-TAGGED INTIMIN AND METHODS OF USING INTIMIN TO STIMULATE ANIMMUNE RESPONSE AND AS AN ANTIGEN CARRIER WITH TARGETING CAPABILITY, ofinventors Marian McKee, Alison O'Brien, and Marian Wachtel ProvisionalApplication No. 60/015,937; filed on Apr. 19, 1996, and ProvisionalApplication No. 60/015,938, filed on Apr. 22, 1996; said applicationsare incorporated herein by reference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, licensed and usedfor governmental purposes without the payment of any royalties to usthereon.

FIELD OF THE INVENTION

This invention relates to a plasmid for engineering plants to expressintimin, alone or as a fusion protein with one or more antigens, and toa method of promoting a protective immune response by the administrationof host organisms transformed with such plasmids. Such protective immuneresponse will be directed to intimin, or portions thereof and/or to theone or more antigens. The host organisms include bacteria, yeast,fungus, and plants.

BACKGROUND OF THE INVENTION

A virulent form of bloody diarrhea is caused by the EnterohemorrhagicEscherichia coli (EHEC). This pathogen is the most common infectiouscause of bloody diarrhea (also called hemorrhagic colitis [HC]) in theUnited States (Centers for Disease Control and Prevention (executivesummary). MMWR 43(No.RR-5):1-18 (1994); Griffin, P. M. et al. Annals ofInternal Med. 109:705 (1988)). One serotype in particular, 0157:H7, isthe most commonly isolated serotype of EHEC in the United States, andhas been linked to a significant number of outbreaks of HC beginning in1982 (Riley, L. W. et al. N.Eng.J. Med. 308:681 (1983)).

The primary mode of transmission of EHEC occurs through ingestion ofcontaminated food, particularly undercooked hamburger (Doyle, M. P. andSchoeni, J. L. Appl. Environ. Microbiol. 53:2394 (1987); Samadpour, M.et al. Appl. Environ. Microbiol. 60:1038 (1994)). Among people infectedby EHEC, as many as 5% suffer a serious complication called HemolyticUremic Syndrome (HUS), a condition caused by the action of Shiga-liketoxins that target and destroy cells lining blood vessels (endothelialcells), such as those present in the glomeruli of the kidney. (Johnson,W. M. et al. Lancet. i:76 (1983); O'Brien, A. D. et al. Lancet. i:702(1983)). HUS can result in permanent kidney damage or even completekidney failure.

Although EHEC can cause very serious illness even in healthy adults,young children in particular are at greater risk of dying or sufferingpermanent damage from the infection. Others for whom the infection canbe particularly dangerous include the elderly and theimmuno-compromised. With the prevalence of EHEC in cattle and thesubjective nature of differentiating between cooked and undercookedhamburger, a convenient stop at a fast food restaurant, or even a familybarbecue, can result in family tragedy.

One key to the deadly nature of EHEC is the bacteria's ability toproduce attaching/effacing (A/E) intestinal lesions in the colon, suchas those demonstrated in gnotobiotic pigs (Tzipori, S. et al. Infect.Immun. 57:1142 (1989)). The A/E lesions demonstrated in pigs arecharacterized by intimate bacterial adherence to the mucosal cells ofthe intestinal lining and dissolution of microvilli (McKee, M. L. et al.Infect. Immun. 63:3739 (1995); Tzipori, S. et al. Infect. Immun. 57:1142(1989)). Similar lesions have been seen in human laryngeal epithelial(HEp-2)(ATCC # CCL23) cells in tissue culture (McKee, M. L. et al.Infect. Immun. 63:3739 (1995); Tzipori, S. et al. Infect. Immun. 57:1142(1989)).

In 1990, Jerse et al. identified a chromosomal gene in a relateddiarrheagenic E. coli strain, Enteropathogenic E. coli (EPEC). Thatgene, designated eae, was found to be required for the bacterium toproduce A/E lesions in tissue culture (Jerse, A. E. et al. Proc. Natl.Acad. Sci. USA. 87:7839 (1990)). The eae gene encoded a 94 kDa outermembrane protein, called Eae, which is the intimin of EPEC. A similarprotein was demonstrated to be present in an EHEC 0157:H7 strain (Jerse,A. E. and Kaper, J. B. Infect. Immun. 59:4302 (1991)).

Recently, investigators demonstrated that intimin is necessary foradherence of EHEC to human epithelial laryngeal (HEp-2) cells and humanileocecal epithelial (HCT-8) cells (ATCC # CCL244) (McKee, M. L. et al.Infect. Immun. 63:3739 (1995)) and for formation of A/E lesions in thepiglet intestine (Donnenberg, M. S. et al. J. Clin. Invest. 92:1418(1993); McKee, M. L. et al. Infect. Immun. 63:3739 (1995)). Althoughhuman studies with EHEC have not been conducted, the intimin proteinfound in EPEC is strongly associated with the production of diarrhea andfever in human volunteers (Donnenberg, M. S. et al. J. Clin. Invest.92:1412 (1993); Levine, M. M. et al. J. Infect. Dis. 152:550 (1985)).

Human volunteers (10 out of 10) challenged with EPEC strain E2348/69mounted a notable immune response to the 94 kDa protein after 28 days(Levine et al. J Infect. Dis. 152:550, 1985)). In these human trials theonly volunteer (1 out of 10) who failed to develop diarrhea afteringestion of E2348/69 was the individual in this group who haddetectable antibody to the 94 kDa protein before challenge.

Two other bacterial species capable of inducing A/E lesions have beenshown to contain the eae locus: Hafnia alvei (Albert, M. J. et al. J.Med. Microbiol. 37:310 (1992)) and Citrobacter freundii biotype 4280(Schauer, D. B., and Falkow, S. Infect. Immun. 61:2486 (1993)). Althoughthese bacteria are not generally associated with pathology in humans,they can cause significant disease in the animal species with which theyare normally associated. For instance, Citrobacter freundii biotype 4280is associated with gastrointestinal illness in mice. Mice often serve ascontrol and test subjects in experiments. Costly and carefullycontrolled experiments can be jeopardized by an outbreak of this diseasein an animal care facility. In addition, such bacterial species maybecome pathogenic to immuno-compromised patients, the young and theelderly.

Animals, such as cows, infected with bacterial strains expressingintimin may become ill themselves, in addition to serving as a source ofsuch infections to others. Eradicating or even limiting these animalreservoirs of intimin-expressing bacteria in animals with antibiotictherapy would be prohibitively expensive. In addition, not only isantibiotic treatment of the infections in humans or animals costly, butthe antibiotics themselves are associated with side effects that can bedangerous. As with EHEC, those side effects can be especially dangerousto young children and the elderly. Consequently, the need exists foranother means of reducing the seriousness of the infections orpreventing them altogether through promotion of protective immuneresponses against bacteria expressing intimin.

A further need is for forms of immunization that are less timeconsuming, expensive and painful than immunization through injection ofantigens. Yet another need is for the generation of protective immuneresponses in the specific tissues involved at the point of infection,most often the gastrointestinal mucosa.

Other organisms infecting gastrointestinal tissue, including, but notlimited to Salmonella sp. and Shigella sp., possess antigens againstwhich an immune response could be generated. A need exists, however, fora means of targeting those antigens to gastrointestinal mucosa, in orderto stimulate a mucosal immune response, as well as stimulatingcirculating antibodies.

Finally, a need exists for alternate means of delivering agents thatpromote a protective immune response.

SUMMARY OF THE INVENTION

The present invention relates to a method of stimulating an immuneresponse comprising transforming a plant with a vector encoding intimin,an intimin-like protein, or a portion thereof, wherein the plantexpresses an intimin, an intimin-like protein, or a portion thereof, andadministering the plant, or a portion thereof, to a patient. The presentinvention also relates to a method of stimulating an immune responsecomprising transforming a plant with a vector encoding intimin, anintimin-like protein, or a portion thereof, wherein said plant expressesan intimin, an intimin-like protein, or a portion thereof, extractingintimin, a portion of intimin, or an intimin-like protein from the plantor portion thereof, administering the extracted intimin, portion ofintimin, or an intimin-like protein to a patient.

The invention additionally relates to a DNA construct that codes for theexpression of a heterologous DNA in a plant, wherein the heterologousDNA encodes intimin, intimin-like protein, or a portion thereof. Theinvention further relates to a plant cell containing a heterologous DNAconstruct that encodes and expresses a heterologous DNA, wherein theheterologous DNA encodes intimin, intimin-like protein, or a portionthereof.

The present invention still further relates to a method of makingtransgenic plant cells, comprising providing a plant cell capable ofregeneration, and transforming the plant cell with a DNA construct asdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts pEB313, a plasmid encoding RIHisEae. This plasmid encodesa histidine-tagged intimin that spans 900 out of 935 predicted aminoacids.

FIG. 2 depicts the predicted protein sequence of the complete EHEC 933eae gene.

FIG. 3 depicts the DNA sequence from EHEC strain CL8, sequenced byBeebakhee, G. et al.

FIG. 4 depicts the DNA sequence from EHEC strain 933, sequenced by Yuand Kaper.

FIG. 5 depicts the 3144 bp fragment of eae produced by PCRamplification, in the region labelled eae.

FIG. 6 depicts pEB311, a plasmid encoding EHEC strain 86-24 eae (entirecoding sequence) driven by the lac promoter.

FIG. 7 depicts pEB310, a plasmid encoding EHEC strain 86-24 eae (entirecoding sequence) driven by the PT7 promoter.

FIG. 8 depicts histidine-tag expression plasmids (Qiagen Inc.) The blackbox marked “6XHis” corresponds to the sequence CAY CAY CAY CAY CAY CAYwhich encodes 6 histridine residues.

FIG. 9 depicts the repressor plasmid (Qiagen Inc.) (multicopy).

FIG. 10 depicts pEB312, a plasmid encoding RVHindHis. This plasmidencodes a histidine-tagged intimin that spans 604 of 935 predicted aminoacids.

FIG. 11 depicts the different fragments of eae cloned into his-taggedvectors, and the corresponding names of these plasmids.

FIG. 12 depicts the different C-terminal fragments of eae cloned intohis-tagged vectors and the corresponding names of these plasmid.

FIG. 13 depicts the construction of an eae mutant, 86-24 eaeΔ10, byallelic exchange.

FIG. 14 depicts pEB290, a plasmid encoding most of the eae structuralgene. The 3′250 bp of eae are not encoded by pEB290.

FIG. 15 depicts pEB300, used to construct the deletion mutant; deletedfor the 1275 bp internal BcI I fragment of eae.

FIG. 16 depicts pAM450, a suicide vector for introduction of clonedgenes into the bacterial chromosome.

FIG. 17 depicts pEB305, a plasmid encoding the deleted eae gene inpAM450 vector for homologous recombination.

FIG. 18 depicts the cloning scheme for construction of a plasmidexpressing N-His-IcsA-intimin-C.

FIG. 19 shows the cloning scheme for construction of a plasmid designedto engineer plants to express his-intimin. The plant expression vectorpKLYX 71S² and the his-intimin encoding plasmid pINT are also depicted.

FIG. 20 shows the cloning scheme for construction of a plasmid designedto engineer bacterial hosts to express his-intimin.

FIG. 21 depicts pGHNC5, a plasmid containing a plant expressioncassette. The MARs are tobacco nuclear scaffold attachment regions. TheClaI-EcoRI fragment containing 3′-rbcs-eae-His-35S² is excised from pINTand ligated into pGHNC5 to create pMAREAE, depicted in FIG. 22.

FIG. 22 depicts the plasmid pMAREAE. The SacI-KpnI fragment containingMAR-35s²-His-eae-3′rbcs-MAR is excised from pMAREAE and ligated intopBIN19 to create pBIN-ME, depicted in FIG. 23. The plasmid pBIN19encodes the NPTII gene (nopaline synthase from Agrebacteriumtumefaciens, conferring kanamycin resistance). NPTII is flanked by anopaline synthase promoter (pNOS), as well as a nopalinesynthase-terminator (3′NOS).

FIG. 23 depicts pBIN-ME, a plasmid containingMAR-35S²-His-eae-3′rbcs-MAR. This plasmid has kanamycin resistanceconferred by the 3′NOS-NPTII-pNOS cassette.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of stimulating an immuneresponse comprising transforming a plant with a vector encoding intimin,an intimin-like protein, or a portion thereof, wherein the plantexpresses an intimin, an intimin-like protein, or a portion thereof, andadministering the plant, or a portion thereof, to a patient. Theinvention further relates to such methods where the intimin,intimin-like protein, or portion thereof additionally comprises at leastone antigen, at least one drug, or a combination thereof,recombinatorially fused to the intimin, intimin-like protein, or portionthereof.

The invention further relates to a method of stimulating an immuneresponse comprising transforming a plant with a vector encoding intimin,an intimin-like protein, or a portion thereof, wherein the plantexpresses an intimin, an intimin-like protein, or a portion thereof,extracting intimin, a portion of intimin, or an intimin-like proteinfrom the plant or portion thereof, and administering the extractedintimin, portion of intimin, or intimin-like protein to a patient. Thisinvention also relates to this method where the intimin, intimin-likeprotein, or portion thereof additionally comprises at least one antigen,at least one drug, or a combination thereof, recombinatorially fused tothe intimin, intimin-like protein, or portion thereof.

The methods described above can also include the step of enriching theintimin, portion of intimin, or intimin-like protein prior toadministration or purifying the intimin, portion of intimin, orintimin-like protein prior to administration. Preferably in theabove-described methods, the intimin is EHEC intimin. It is alsopreferred that the intimin, intimin-like protein, or portion thereoffurther comprises a histidine tag.

In the above-described methods, the plant can be monocotyledonous ordicotyledonous. Examples of such plants are described in more detailbelow, but more preferably include alfalfa, carrot, canola, tobacco,banana, and potato plants.

The present invention also relates to a DNA construct that codes for theexpression of a heterologous DNA in a plant, wherein the heterologousDNA encodes intimin, intimin-like protein, or portion thereof. Theheterologous DNA additionally can also code at least one antigen, atleast one drug, or a combination thereof, recombinatorially fused to theintimin, intimin-like protein, or portion thereof.

Preferably the DNA construct is a plant transformation vector. The planttransformation vector is preferably Agrobacterium vector. It is alsopreferred that the plant transformation is conducted via amicroparticle. It is additionally preferred that the planttransformation vector is a viral vector.

In the above-described DNA constructs according to the invention,preferably the intimin, intimin-like protein, or portion thereof encodedby said DNA further comprises a histidine tag. It is also preferred thatthe codons of the heterologous DNA are replaced with codons that arepreferred for expression in a plant cell.

The present invention is still further directed to a plant cellcontaining a heterologous DNA construct that encodes and expresses aheterologous DNA, wherein the heterologous DNA encodes intimin,intimin-like protein, or a portion thereof. In such plant cells, theheterologous DNA can additionally encode at least one antigen, at leastone drug, or a combination thereof, recombinatorially fused to theintimin, intimin-like protein, or portion thereof. Preferably in theseplant cells the intimin, intimin-like protein, or portion thereofencoded by the DNA further comprises a histidine tag.

The present invention still further yet relates to a method of makingtransgenic plant cells, comprising providing a plant cell capable ofregeneration, and transforming the plant cell with a DNA construct asdescribed above. Preferably the transforming step of this method iscarried out by infecting the plant cell with an Agrobacterium vectorthat transfers the DNA construct into the plant cell. It is alsopreferred that the transforming step is carried out by bombarding theplant cell with microparticles carrying the DNA construct. Additionally,it is preferred that the transforming step is carried out bytransformation with a viral vector.

Preferably for the above-described methods, the plant cell resides in aplant tissue capable of regeneration. The above-described method canalso comprise the step of regenerating shoots, roots, or a plant fromthe transformed plant cells. In this method, the plant preferably ismonocotyledonous or dicotyledonous. It is also preferred that, for thismethod, the intimin, intimin-like protein, or portion thereof encoded bythe DNA further comprises a histidine tag.

An object of the invention is to express intimin in a host organism,which host will be administered or fed, directly or after processing, toanimals or humans in order to stimulate an immune response to intimin.Such an immune response will protect the animal or human againstillness, disease and related sequelae caused by EHEC and other pathogenshaving the capacity to bind epithelial cells through proteins havingsome degree of homology with the specific intimin expressed by EHEC.

The object is achieved through stimulation of an immune responsedirected against intimin, thereby blocking the capability of EHEC toadhere to epithelial cells. Consequently, the term immunization is usedin the application. The degree of protection will vary with the degreeof homology with intimin as well as the unique attributes of thepatient, particularly as different species will be treated. The precisedegree of protection is unimportant to quantitate in practicing theinvention for any particular pathogen. In addition, the inventiondescribes the expression of intimin in a host organism as a fusionprotein with one or more other antigens and administration of hostorganism to promote a protective immune response against intimin and theone or more antigens.

In addition to stimulating an immune response that permits a patient toavoid infection altogether, immunization as used herein also meansdecreasing the ability of the pathogens to colonize the gastrointestinaltract and decreasing the severity of an infection, as measured by any ofthe following indicators: reduced incidences of death, HUS, or permanentkidney damage; decreased levels of toxins; reduced fluid loss; or otherindicators of illness regularly used by those ordinarily skilled in therelevant art. Thus, an immuno-protective amount is that amountsufficient to decrease the ability of the pathogens to colonize thegastrointestinal tract as well as decreasing the severity of aninfection as measured by the above indicators.

A preferred host of the invention is a plant cell.

Plant cells have successfully been engineered to express heterologousgenes, such as those of bacterial origin. Crop plants of all types havebeen engineered with genes from bacterial origin. Some examples of theseare the commonly-used antibiotic resistance genes, such as the scorablemarker genes for neomycin phosphotransferase (NPTII) and hygromycinphosphotransferase (HPII). These genes were isolated from the bacteriumE. coli (Fraley, R. T., et al., Proc. Natl. Acad. Sci. USA 80: 4803(1983); Vandenberghe et al., Plant Mol. Biol. 5: 299 (1985)). Anotherpopular scorable marker gene routinely used in plant transformationstudies also comes from E. coli: beta glucuronidase (GUS) (JeffersonPlant Mol. Biol. Rep. 5: 387-405 (1988)). All of these genes have beenuseful and have been highly expressed by transgenic plants, i.e., thosecontaining heterologous DNA, in their native form; they required nomodifications in their coding sequence.

Other genes from bacteria, however, have been poorly expressed whenengineered into plants. One example is the mercuric ion reductase genefrom E. coli Rugh, C. L.; Wilde, H. D.; Stack, N. M.; Summers, A. O.;and Meagher, R. B.; Mecuric ion reduction and resistance in transgenicArabidopsis thaliana plants expressing a modified bacterial merA gene.Proc. Natl. Acad. Sci. USA 93(8): 382-87 (1996). It requiredmodification in its coding sequence before it could be expressed.Perhaps the best-known examples are insecticidal cry genes from Bacillusthuringiesis. They have all exhibited low to no expression until theywere “rebuilt” or codon optimized for expression in plants (Perlak etal., Proc. Natl. Acad. Sci. USA 88: 3324-3328 (1991); Adang et al.,Plant Mol. Biol. 21: 1131-1145 (1993)). In these studies, researchersreconstructed the genes by synthesizing and linking oligonucleotidesthat encode preferential codons for the plant species, without changingthe amino acid sequence. By matching the codon usage of the new gene toplant-preferred codons, the introduced gene can be highly expressed(e.g., Stewart et al., Insect control and dosage effects in transgeniccanola, Brassica napus L. (Brassicaceae), containing a syntheticBacillus thuringiensis Cryla(c) gene. Plant Physiology, 112:115-120(1996)). Thus, the expression of bacterial genes by plant cells has beenaccomplished.

Plants engineered with a foreign gene have been successful deliveryagents for oral vaccines. As set forth in a recent review, (Mason andArntzen, Tibtech 13: 388-392 (1995)), the art has recognized such usesof engineered plants. The body of work also includes the recentdemonstration that, when expressing genes that code for antigens ofviral and bacterial pathogens in plants, the antigens retain theirimmunogenic properties (Mason and Arntzen, Tibtech 13: 388-392 (1995)).Mason et al. (Mason et al, Proc. Natl. Acad. Sci. USA 89: 11745-11749(1992)) introduced the concept of engineering plants as a vehicledelivery system for vaccines and have since shown that their system iseffective for hepatitis B (Thanavala et al., Proc. Natl. Acad. Sci. 92:3358-3361 (1995)), E. coli enterotoxin B subunit and cholera-toxin Bsubunit (Haq et al., Science 268: 714-716 (1995)). One basis for theeffectiveness of this strategy rests on the fact that the antigensstimulate mucosal immunity.

In the practice of this invention, a fragment of the intimin gene, eae(which may, for example, contain the his tag, such as the XhoI-HindIIIfragment of pEB313) is ligated to a plant promoter in an appropriatevector. The introduction of this vector in, for example, tobacco plantsby appropriate methods results in the expression of intimin, such ashis-intimin, by the tobacco plants. Once the tobacco plants are grown,they are homogenized to make a “tobacco soup” (protein extract). Thissoup is then used as an adsorbent for an ELISA, using standardmethodology, to detect the presence of intimin. Alternatively, thisextract is run on an SDS-PAGE gel for Western blot analysis. One can usepolyclonal antisera directed against the intimin or, to detect thepresence of a histidine tag, antibody directed against the his tag(available from QIAGEN) for such analysis. The amount of intiminexpressed from the plant can be quantitated using the ELISA or Westernblots.

Using a similar approach, a skilled artisan may express intimin, or forexample intimin as a fusion protein with one or more other antigens, inthe tobacco plant or other plants, such as carrots, bananas, canola, andalfalfa, although other plants are within the scope of the invention.When such transformed plants are fed to animals, such as cattle thetransformed plants express the intimin protein or the fusion protein,thereby delivering the antigens to the animals, in order to stimulate animmune response. Such a method is desirable in that it is an inexpensiveand efficient method for protecting the animal and, moreover, protectingagainst future colonization of such animals by human pathogens such asEHEC reduces the risk of infection for humans.

In one transformation procedure, the eae gene is put under the controlof a constitutive plant promotor in an Agrobacterium tumefaciens binaryvector and the plants are engineered by Agrobacterium-mediatedtransformation.

The intimin expressed in the host organism is of a size that retainsbinding function, and may include intimin that has been histidinetagged. In addition, the intimin may be expressed in the host as afusion protein with one or more other antigens. Administration of thehost to patients, including animals, stimulates an immune response tothe intimin and the one or more antigen.

Those skilled in the art will also recognize that the size of thehis-tagged intimin to be used may be varied according to the specificpurpose for administering the intimin. For example, if the his-taggedintimin is to be conjugated with one or more antigens, a smallerfragment may be selected to enhance stability of the combined fusionproduct; although the use of a larger fragment is by no means precluded.The size or conformation of the other antigen and the location of itssignificant epitopes may indicate that a particular length of his-taggedintimin will bring the epitopes into close proximity to the mucosa. Thedesired size will also vary with the convenience of availablerestriction sites, in light of the materials and methods known to thoseof ordinary skill in the art. Consequently, the terms his-intimin andhis-tagged intimin, as used herein, mean polypeptides containing atleast 900 out of the 935 predicted C-terminal amino acids of intiminwith a histidine tag (full-length intimin) and smaller portions of thehis-tagged protein that retain binding function.

The conjugate may be generated through recombinant technology togenerate a fusion protein comprising a portion of intimin and at leastone additional protein antigen or drug. In addition, intimin may bechemically or physically conjugated to drugs, proteins, peptides,carbohydrates and other antigens by methods known to those skilled inthe art. Methods of chemical conjugation are well known to those skilledin the art, and include, in part, coupling through available functionalgroups (such as amino, carboxyl, thio and aldehyde groups). See S. S.Wong, Chemistry of Protein Conjugate and Crosslinking CRC Press (1991);and Brenkeley et al. Brief Survey of Methods for Preparing ProteinConjugates With Dyes, Haptens and Cross-linking Agents, BioconjugateChemistry 3 #1 (Jan. 1992).

The retention of binding function means that the intimin, intimin-likeproteins, and/or portions thereof retain the capacity to bind toepithelial cells or cell lines, such as HEp-2 and HCT-8. Such bindingmay be visualized as bacterial microcolony formation by the microcolonyassay (Frankel et al., Infect. Immun. 62(5):1835-1842 (1994)), by FAS,(florescence actin staining), or both (McKee and O'Brien, Infect. Immun.63(5):2070-2074 (1995), the disclosures of which is incorporated byreference herein). Additionally, binding may be measured by any methodstandard in the art including in vivo measurement as a function ofbacterial virulence-or pathogenicity, or by post-mortem histologicalexamination. Examples of each of the above methods for determiningbinding function are detailed in Examples below, including the adherenceassay described in Example IV.

Unless specified otherwise, the uses and methods set forth herein aregenerally applicable to humans and animals. The term patient is usedherein to mean both humans and animals, and animals is not limited todomesticated animals but also may include wildlife and laboratoryanimals as well.

Isolating and Purifying His-tagged Intimin

It has been shown that a His-intimin fusion, in which the N-terminalthird of the molecule is deleted (RVHindHis), was capable ofcomplementing adherence of a non-adherent EHEC eae mutant; i.e.,restored binding capability to a strain of EHEC that lost its bindingcapability following genetic alterations that prevented expression ofintimin. The pattern of adherence demonstrated in the restored bindingwas indistinguishable from that observed in wild-type strain 86-24(McKee, M. L. and O'Brien, A. D. Infect. Immun. In press (1996)). Wheremeasured by the microcolony assay, described above, wild-type activityis identified as a punctate pattern with localized areas of intensestaining.

Purification of the intimin protein, however, was difficult, in partbecause intimin is always associated with the outer membrane of EHEC.The majority of the overexpressed recombinant intimin remainedassociated with the bacterial membrane fraction, even after sonicdisruption of the host bacterium and addition of mild detergent to theextraction buffer. The insolubility of the intimin protein, combinedwith the abundance of other native E. coli proteins in the 97 kDa range,made purification of the native protein difficult.

Attempts to make an intimin-maltose binding protein fusion (MBP) alsowere unsuccessful because the predicted protein product had deletionsand rearrangements. An attempt by other investigators had shown aconstruct of MBP fusions to the C-terminal 280 amino acids of intimin,but the fusion did not confer EHEC-like binding function. The diffusepattern of adherence conferred by the MBP-intimin fusion protein wasclearly different from the pattern of EHEC binding to HEp-2 cells(Frankel, G. et al. Infect. Immun. 62:1835 (1994)).

One possible explanation for the failure to obtain a functionalMBP-intimin fusion larger than 280 amino acids is that overexpression ofa piece of Eae greater than the last 280 amino acids is unstable, andthus prone to rearrangements, i.e. it may be impossible to isolate thatclone because it is lethal or deleterious to the cell when expressed.Alternatively, overexpression of a piece of MBP-intimin fusion largerthan 280 amino acids could plug up the bacterial membrane, which wouldbe lethal to the cells.

After trying unsuccessfully to purify intimin using MBP, a fusion wascreated using the QIAEXPRESSIONIST® Kit (Qiagen, Inc., 6xHis taggedprotein constructs purified on nickel-nitrilotriacetic acid (Ni-NTA)metal affinity chromatography), which involves attaching a histidine tagto the protein. The histidine tag binds tightly to a nickel affinitymatrix, which facilitates purification of large quantities of materialfor further studies. In addition, the expression system permits one tomaintain tight control of expression of the His fusion proteins toprevent any possible lethal effects of the recombinant protein on the E.coli host strain as a result of overexpression of the protein. Example Idescribes the creation of such a fusion protein.

EXAMPLE I

A. Construction of a Plasmid, pEB313 (FIG. 1), Encoding His-taggedIntimin Encompassing 900 Out of 935 Predicted Amino Acids (FIG. 2)

The eae gene is cloned from EHEC strain 86-24 (serotype 0157:H7),readily obtainable from Griffin, P. M. et al., Ann. Intern. Med.109:705-712 (1988), or Phil Tarr (Children's Hospital and MedicalCenter, 4800 Sand Point Way NE, Seattle, Wash. 98105, 206-526-2521.) TheDNA is extracted according to standard chromosomal prep techniques(Wilson, K. in Current Protocols in Molecular Biology, Ausubel, F. M. etal. (eds.) vol 1:2.4.1—“Preparation of Genomic DNA from Bacteria”).

The gene is cloned using the polymerase chain reaction (PCR), a standardtechnique in the art, using primers designed from a composite of the 2known EHEC eae sequences. Two primers are constructed:Sn20-CGTTGTTAAGTCAATGGAAAC(SEQ ID NO:1) a 5′ primer, spanning bases20-41 of the sequence from the strain CL8, which was sequenced byBeebakhee, G. et al. FEMS Microbiology. 91:63 (1992) (FIG. 3); andMM2-TCTAGAGAGAAAACGTGAATGTTGTCTCT(SEQ ID NO:2), a 3′ primer, spanningbases 3061-3082 of the sequence from strain 933, sequenced by Yu, J. andKaper, J. B. Mol. Microbiol. 6:411 (1992) (FIG. 4). MM2 was furtherdesigned to include an XbaI site at the 3′ end.

The PCR reactions are performed using a PCR reagent kit utilizingAMPLITAQ®, or other DNA polymerase, according to the instructions of themanufacturer, Perkin Elmer. The PCR amplification produces a 3144 bpfragment encoding the entire eae open reading frame (ORF) (FIG. 5,region designated eae) and includes 186 bp upstream. The PCR product isprocessed to create blunt ends and ligated into the EcoRV site of thevector pBRKS (Schmitt et al., J. Bacteriol. 176:368-377 (1994)).

The gene is cloned in both directions to allow transcription from eitherP_(lac) pEB311 (FIG. 6) and from P_(T7) pEB310 (FIG. 7) under theappropriate conditions. The plasmids are transformed into host strainXL1BlueF′Tn5 lacl_(Q), (available from QIAGEN, Inc., 9600 DeSoto Ave.,Chatsworth, Calif. 91311, 1-800-362-7737). The recombinants aremaintained under the constitutive control of the lac repressor, becausethe previous failed attempts to clone eae suggested that overexpressionof eae might be lethal to the host E. coli strain. The lower copy numberof pBRKS vector, along with the control conferred by the lac repressorobviates these problems.

A his-tagged intimin plasmid is constructed by digesting pEB310 withEcoRI, filling in with Klenow fragment, digesting with HindIII, andisolating the resulting 2895 bp fragment. The DNA fragments are isolatedusing the GENECLEAN®, an agarose gel DNA purification procedure (BIO101, Inc. (1070 Joshua Way, Vista, Calif. 92083, 1-800-424-61010). Thehis-tag expression plasmid pQE32 (FIG. 8) (available from QIAGEN, Inc.)is digested with SmaI and HindIII. The 2895 bp fragment is then ligatedto pQE32, creating pEB313 (FIG. 1).

This plasmid, pEB313, encodes a his-tagged Eae fragment of 101 kDa,called RIHisEae, which encodes 900 out of 935 predicted amino acids.This his-intimin fusion is constructed so that the N terminal 35 aminoacids are deleted, to remove any potential signal sequence. A signalsequence could target the fusion protein to the membrane or lead tocleavage of the His tag from the encoded intimin.

After the pEB313 construct is made, it is transformed into a lab strainof E. coli containing the lac repressor (lacl_(Q)), such as M15 pREP4(repressor contained on the multicopy plasmid pREP4, supplied by QIAGEN,Inc.) (FIG. 9) or XL1 BlueF′Tn5lacl_(Q) (repressor contained on thesingle copy F′ plasmid, also available from QIAGEN, Inc.). Thetransformed E. coli express the his-intimin fusion protein encoded bypEB313. Purification of the protein is accomplished as set forth inExample III.

B. Construction of a Plasmid, pHis-Inv1, Encoding His-tagged Yersiniapseudotuberculosis invasin, and Construction of a Plasmid, pHis-Inv2,Encoding His-tagged Yersinia pseudotuberculosis invasin with a Deletionof the N-terminal 40 Amino Acids

The plasmid pRI203 contains a 4.6 kb fragment of Yersiniapseudotuberculosis chromosomal DNA, including the inv gene andsurrounding nucleotides sequences, and is readily obtainable from Dr.Ralph Isberg (Dept. Of Molecular Biology and Microbiology, TuftsUniversity School of Medicine, Boston, Mass. 02111; ref. R. R. Isberg,D. L. Voorhis, and S. Falkow. Cell. 50:769 (1987)). The pRI203 DNA isextracted from the supplied bacterial strain with the use of a QIAGENDNA extraction kit (QIAGEN, Chatsworth, Calif.) according to theinstructions of the manufacturer.

To construct pHis-Inv1, two primers, Inv1 (=5′GTACGGATCCATGATGGTTTTCCAGCCAATCAGTGAG 3′(SEQ ID NO:3) and Inv3 (=5′GTACGGTACCTTATATTGACAGCGCACAGAGCGGG 3′(SEQ ID NO:4)) are used in a PCRreaction (as described above in part A) to amplify the desired invsequences from pRI203. The resulting 2960 bp inv fragment is digestedwith BamHI and KpnI, run on an agarose gel, excised with a razor, andpurified using GENECLEAN® (Bio101, LaJolla, Calif.). The purified invfragment is ligated into the His-tag QIAGEN vector containing theappropriate reading frame corresponding to the amplified inv sequence(pQE30, 31 or 32, QIAGEN, Chatsworth, Calif.), digested with BamHI andKpnI. The ligated plasmids are then transformed into DH5αF′Tn5lacl_(Q),and transformants checked for the presence of the appropriate sizeinsert.

The plasmid pHis-Inv2 is constructed in the event that a signal sequence(and therefore the adjoining His tag) is cleaved from the proteinexpressed from pHis-Inv1. To construct pHis-Inv2, two primers, Inv2 (=5′GTACGGATCCATATGTGGAATGTTCATGGCTGGGG 3′(SEQ ID NO:5)) and Inv3 (=5′GTACGGTACCTTATATTGACAGCGCACAGAGCGGG 3′(SEQ ID NO:4)) are used in a PCRreaction (as described in part A above) to amplify the desired invsequences from pRI203. The resulting 2840 bp inv fragment is purified asabove, and ligated into the His-tag QIAGEN vector as above, andtransformed into a similar bacterial host strain.

Alternatively, similar plasmids are constructed by restriction enzymedigestion of pRI203, followed by ligation into the QIAGEN His-tag vector(pQE30, 31 or 32) containing the appropriate reading frame relative tothe 5′ end of the invasin fragment. The preceeding examples are meant toillustrate the construction of plasmids encoding histidine-taggedinvasin from Yersinia pseudotubercuis. One of ordinary skill in the artrecognizes that analogous constructs, designed using alternative vectorsand/or other intimin-like proteins can be constructed according tostandard methods in the art. One of ordinary skill in the art alsorecognizes that the above his-tagged invasins and other his-taggedintimin-like proteins may be applied to the methods disclosed elsewhereherein.

C. Construction of a Plasmid (pEB312) (FIG. 10) Encoding His-taggedIntimin Encompassing 604 out of 935 Predicted Amino Acids

Digested plasmid pEB310 (obtained as described in part A) with EcoRV andHindIII, isolating the 1971 bp fragment, and ligating the fragment intopQE32 digested with SmaI and HindIII. Restriction enzymes, ligases,Klenow fragment used in protocols are from New England BioLabs (32 TozerRd., Beverly, Mass. 01915-55991, 1-800-NEB-LABS) or Gibco BRL (P.O. Box681 Grand Island, N.Y. 14072-0068, 1-800-828-8686). The resultingplasmid is designated pEB312.

The plasmid pEB312 encodes a his-tagged Eae fragment called RVHindHis,which is about 65 kDa and encodes 604 out of 935 predicted amino acids.This construct contains the C-terminal two-thirds of the wild-typeintimin protein.

As with the pEB310-expressed intimin, the fusion proteins remainprimarily in the insoluble pellet after sonic disruption of the host E.coli. Therefore, urea and guanidine HCl are included in the purificationprotocol, which allows extraction of the fusion proteins from theinsoluble pellet.

D. Construction of Additional Plasmids Expressing Different Fragments ofeae

Each of these plasmids is tested to ensure that the protein fragmentpossesses full binding function, using an adherence assay. (See exampleIV, below). In addition, the selection of the size of the intimin varieswith whether the protein is expressed alone and whether cross-immunityis desired with an intimin-like protein of known amino acid sequence. Inaddition to intimin expressed from EHEC and EPEC, examples ofintimin-like proteins include, but are not limited to, intimin-likeproteins of Citrobacter rodentium, Hafina alveii, and the invasins ofYersinia enterocolitica and Yersinia pseudo tuberculosis.

For instance, the larger 900 aa intimin is selected if there isheightened desire for cross-immunity with Yersinia pseudotuberculosis,because EHEC intimin shares 31 % identical and 51% similar amino acidswith the protein of Yersinia pseudotuberculosis, known as invasin (Yu,J. and Kaper, J. B. Mol. Microbiol. 6:411 (1992)). A greater degree ofhomology exists within the amino terminal two-thirds of the respectiveproteins.

Invasin is a 103 kDa outer membrane protein that allows bacterialpenetration of cultured epithelial cells (Isberg, R. R. et al. Cell.60:769 (1987)) and efficient penetration of the intestinal epithelium invivo (Pepe. J. C. and V. L. Miller. Proc. Natl Acad. Sci. USA. 90:6473(1993)). Cell adhesion receptors on the intestinal epithelium, whichhave been identified as integrins, bind invasin prior to bacterialinternalization (Isberg, R. R. and J. M. Leong. Cell. 50:861 (1990)).The central portion of invasin is responsible for protein localizationto the outer membrane, while the C-terminus is required for receptorbinding (Isberg, R. R. Mol. Microbiol. 3:1449 (1989)).

Similarly, a larger 900 aa intimin is selected if there is heighteneddesire for cross-immunity with Enteropathogenic Escherichia coli (EPEC),Citobacter rodentium, or Hafnia alvei. EPEC intimin shares 83% identitywith EHEC intimin over the entire length of the protein; the N-terminal75% of the proteins share 94% identity, while the C-terminal 25% of theproteins share 49% identity (Yu, J. and Kaper, J. B. Mol. Microbiol.6:411 (1992)). Citrobacter freundi intimin shares 84% nucleotideidentity to EHEC eae with the first 2106 bp, and 57% identity to the 3′702 bp (Schauer, D. B. and Falkow, S. Infect. Immun. 61:2486 (1993)).

The plasmids that express different fragments of eae can be constructedusing one of the following techniques: (1) use of a convenientrestriction site within eae, for example, pEB313 digested with SaI andHindIII, isolation of the 1298 bp fragment, and ligation to pQE30(QIAGEN, Inc.) digested with SmaI and HindIII. The resulting plasmidexpresses the last 432 amino acids at the C terminus of Eae; (2)deletion of the 5′ or the 3′ region of any desired fragment usingnuclease BAL-31, Exonuclease III, or Mung bean nuclease (all availablefrom New England BioLabs, 32.Tozer Rd., Beverly, Mass. 01915); (3)construction of plasmids with noncontiguous eae fragments, also usingthe above techniques; and (4) construction of plasmids encoding desiredspecific sequences with the use of PCR primers specifying the 5′ and 3′ends of such sequences, as described in greater detail below.

With respect to the third technique, a His-tagged middle third intimindeletion mutant plasmid is constructed (pMW114). The plasmid pMW106(described below) is transformed into the dam strain DM1F′Tn5/ac/_(Q).DNA is made using the QIAGEN kit (QIAGEN, Inc. an alkaline lysis/anionexchange chromatography plasmid purification system), and is digestedwith BcA/I. The DNA is runout on an agarose gel. The 5178 bp band is cutout, is purified using GENECLEAN® (Bio 101), is ligated, and thentransformed into DH5αF′Tn5ac/_(Q) (or other appropriate strain such asXL1 Blue or M15pREP4). Transformants are checked by restrictiondigestion of DNA. This description does not preclude the construction ofother plasmids encoding non-contiguous eae sequences.

With respect to the fourth technique, PCR can be used to amplifyspecific fragments of eae; the fragments are isolated by restrictionenzyme digestion and agarose gel electrophoresis, and ligated into theappropriate His-tag expression vector (i.e. p.QE30, 31 or 32; QIAGEN,Inc.).

For example, clones are constructed encoding various regions of eae (seeFIGS. 11 and 12). The capacity of these clones to retain adherencefunction is assessed by either (1) transformation into the eae mutant,followed by adherence assays with HEp-2 cells (see Ex III, section C),or (2) addition of exogenous protein to bacteria (see Ex III, sectionD). It is important that the fragment selected retains full or as closeto full wild type binding activity.

It is hypothesized that clones containing the highest binding activitywill include the C-terminal third (third third) of the protein, perhapsas little as 150 C-terminal amino acids. This hypothesis is supported,for example, by the findings disclosed by Frankel et al., Infection &Immunity, 62:1835 (1994), Frankel et al., Infection & Immunity, 63:4323(1995), and Frankel et al., J. Biol. Chem., 271:20359 (1996). Proteinscannot be thought of as linear arrays of amino acids; rather they existin a 3-dimensional structure. It is important to keep in mind that asingle amino acid change or a deletion of a portion of the protein canperturb this structure. Therefore, full cell binding activity mayrequire the presence of additional non-contiguous sequences along withthe third third putative binding domain.

It is further hypothesized that clones containing high binding activitywill include the two C-terminal Cys (encoded at bp 2780 and bp 3002,numbering ref; Beebakhee, G., J. DeAzavedo, and J. Brunton, FEMSMicrobiology Letters 91:63 (1992)) for hypothesized disulfide bondformation and resulting loop formation. Additionally, it is hypothesizedthat clones containing high binding activity may require one or bothaspartate(s) (encoded at bp 2819 and 2828, numbering ref. Beebakhee).This hypothesis is supported, for example, by analogy to invasin, asdesribed in Leong, J. M., Embo. J. 14:422 (1995).

All clones are constructed in a similar manner. PCR primers are designedwhich specify the 5′ and 3′ region of the desired eae fragment. Tofacilitate cloning into pQE31, each 5′ primer (MW1, MW3, MW5, MW7, MW8,MW9, MW10) contains a 5′ BamHI site, and each 3′ primer (MW2, MW4, MW6,MW11, MW12) contains a 5′ KpnI site. Each PCR primer is designed so thatthe reading frame of the specified eae sequence is appropriate forinsertion into pQE31. The following His-tagged constructs are clonedusing the indicated PCR primers:

(1) pMW101—encodes the N-terminal third of Eae; 27 kDa protein (PCRprimers: MW1 (5′ PCR primer)=5′ GTACGGATCCGAATTCATTTGCAAATGGTG 3+(SEQ IDNO:6); MW2 (3′ PCR primer)=5′ GTACGGTACCTGATCMTGAAGACGTTATAG 3(SEQ IDNO:7));

(2) pMW102—encodes the middle third of Eae; 42 kDa protein (PCR primersMW3 (5′ PCR primer)=5′ GTACGGATCCTGATCAGGATTTTTCTGGTG 3′(SEQ ID NO:8);MW4 (3′ PCR primer)=5′ GTACGGTACCTGATCAAAAAATATAACCGC 3′(SEQ ID NO:9));

(3) pMW103—encodes the C-terminal third (282 amino acids) of Eae; 32 kDaprotein (PCR primers: MW5 (5′ PCR primer)=5′GTACGGATCCTGATCAAACCAAGGCCAGCATTAC 3′(SEQ ID NO:10); MW6 (3′ PCRprimer)=5′ GTACGGTACCTTATTCTACACAAACCGCATAG 3′(SEQ ID NO:11));

(4) pMW104—encodes the N-terminal two thirds of Eae; 69 kDa protein (PCRprimers: MW1 and MW4);

(5) pMW105—encodes the C-terminal two thirds of Eae; 73 kDa protein (PCRprimers: MW3 and MW6);

(6) pMW106—encodes Eae with a small N-terminal 35 amino acid deletion;100 kDa protein (PCR primers: MW1 and MW6);

(7) pMW108—encodes the C-terminal 150 amino acids of Eae (PCR primers:MW7 (5′ PCR primer)=5′ GTACGGATCCACTGAAAGCGCGGTGGTGATB 3′(SEQ ID NO:12);MW6);

(8) pMW109—encodes the C-terminal 140 amino acids of Eae (PCR primers:MW8 (5′ PCR primer)=5′ GTACGGATCCTTCATGGTATTCAGAAAATAC 3′(SEQ ID NO:13);MW6);

(9) pMW110—encodes the C-terminal 130 amino acids of Eae (PCR primers:MW9 (5′ PCR primer)=5′ GTACGGATCCGACTGTCGATGCATCAGGGAAAG 3′(SEQ IDNO:14); MW6);

(10) pMW111—encodes the C-terminal 120 amino acids of Eae (PCR primers:MW10 (5′ PCR primer)=5′ GTACGGATCCGAATGGTAAAGGCAGTGTCG 3′(SEQ ID NO:15);MW6);

(11) pMW112—encodes 120 amino acids of Eae with the C-terminus, spanningbp #2560-2923, (numbering refers to eae sequence of strain CL8 ref.Beebakhee, G., J. DeAzavedo, and J. Brunton. FEMS Microbiology Letters91:63)). (PCR primers: MW7; MW11 (3′ PCR primer)=5′GTACGGTACCTCCAGAACGCTGCTCACTAG 3′(SEQ ID NO:16));

(12) pMW113—encodes the C-terminal 282 amino acids of Eae, Cys at bp3002 changed to Ser with the use of the PCR primer MW12 (numberingrefers to eae sequence of strain CL8 ref. Beebakhee, G., J. DeAzavedo,and J. Brunton. FEMS Microbiology Letters 91:63 (1992)) (PCR primers:MW5; MW12 (3′ PCR primer)=5′ GTACGGTACCTTATTCTACAGAAACCGCATAG 3′(SEQ IDNO:17)).

All clones are constructed by first diluting lyophilized primers to 10μM with dH₂O. Template DNA from strain XL1 blue pEB310 (encoding theentire eae gene) is made using a QIAGEN prep (QIAGEN, Inc.), islinearized by digestion with a restriction enzyme that does not cutwithin or near the coding region, for example HindIII, and isquantitated using a spectrophotometer. PCR reactions are conducted bycombining 10 μl 10×Taq buffer (Perkin Elmer/Roche, Branchburg, N.J.), 10μl 2 mM dNTP mix (Boehringer Mannheim, Indianapolis, Ind.), 10 μl 10 μM5′ PCR primer, 10 μl 10 μM 3′ PCR primer, 6 μl 25 mM MgCl₂ (PerkinElmer/Roche), 52 μl dH₂O, and 1 μl (1-10 ng) linear template DNA. Twodrops of mineral oil are applied to the mixture, which is heated to 100°C. for 5 minutes to denature the template. One μl (5U) of AMPLITAQ®polymerase (Perkin Elmer/Roche) is added, and the PCR reactions arebegun: 95° C./1 min, 50° C./ 1 min, 72° C. 3 min for 30 cycles, followedby 72° C./10 min, and holding at 4° C. After the PCR reactions arecompleted, the DNA is applied to a WIZARD® (Promega, Madison Wis.), aresin based PCR clean up kit, and resuspended in 50 μl TE buffer. PCRamplified DNA is digested with BamHI and KpnI, electrophoresed on anagarose gel, the appropriate size band cut out, and purified byGENECLEAN® (Bio101, Lajolla, Calif.). Digested PCR fragments are thenligated into pQE31 digested with BamHI and KpnI, transformed intoDH5αF′Tn5/ac/_(Q) (or other appropriate strain, such as M15pREP4 orXL1Blue), and transformants checked for the presence of the appropriatesize insert.

With respect to any of the above-listed techniques, if it is necessaryto later remove the Histidine tag from the purified protein, a proteasecleavage site can be inserted between the 6XHis sequence and theN-(N-terminal tag) or C-terminus (C-terminal tag) of the protein. Forexample, Enterokinase recognizes the sequence “DDDK(SEQ ID NO:26)”(Asp₄-Lys), and cleaves after the lysine. A PCR primer encoding thissequence is designed and used to perform site-directed mutagenesis ofthe desired gene fragment. Alternatively, Carboxypeptidase A can be usedfor the removal of C-terminal His tags. This enzyme efficiently removesaromatic C-terminal residues (Hoculi, E. Chemische Industrie. 12:69(1989)) until it encounters a basic residue, at which point removal isterminated. Additionally, PCR can be used to design a primer so that theprotease site is encoded at the N- or C-terminus of the protein encoded;or PCR can be used to design the vector including those sites, and theabove-techniques can be used to clone into the aforementioned vector.

All fragments of intimin expressed from pEB312 or other constructs arepurified using a protocol similar to the protocol detailed in ExampleII, for large scale purification of intimin. It is apparent that thoseof ordinary skill in the art may select additional restriction sites ormodify the protocol while remaining within the scope and spirit of theinvention.

EXAMPLE II

Large Scale Enrichment of Histidine-tagged Intimin

Growing Large-scale Expression Cultures

Inoculate 20 ml LB (Luria-Bertaini) broth containing 100 μg/mlampicillin and 40 μg/ml kanamycin with a loopful of M15 pREP4 pEB313(prepared as described in Example I, above). Grow overnight (15-18 h) at37° C., shaking vigorously. Inoculate 1L of LB broth containing 100μg/ml ampicillin and 40 μg/ml kanamycin with 20 ml of the overnightculture. Grow culture at 37° C. with vigorous shaking until theOD₆₀₀=0.7-0.9 (˜3 h). Add IPTG (isopropyl β-D-thiogalactopyranoside,Sigma Chemical Co., P.O. Box 14508, St. Louis, Mo. 63178,1-800-325-3010) to a final concentration of 1 mM (0.476 g) and continueto grow culture for another 3 h. Divide supernatant into 500 ml bottles(previously weighed) and centrifuge at 4000×g for 10 minutes. Discardthe supernatant, weigh cell pellet, and store at −7° C., or processimmediately.

Thaw cells for 15 minutes, vortex and resusupend in Buffer A [6 M GuHCl,0.1 M NaH₂PO₄, 0.01 M Tris-HCl, pH 8.0] at 5 ml/g wet weight. Stir cellsfor 1 hour at room temperature. Centrifuge lysate at 10,000×g for 15min, collect supernatant. Add 5 ml of a 50% slurry of Ni-NTA resin (Ni-NTA slurry from QIAGEN, Inc), previously equilibrated with Buffer A.Stir at room temperature for 45 minutes, let the slurry settle, removethe supernatant, add 5 ml Buffer A, let the slurry settle, remove thesupernatant, add 5 ml Buffer A, and load the resin into a column. Thecolumn is washed with 10 column volumes of Buffer A, followed by washeswith Buffer B [8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-HCl, pH 8.0] untilthe OD₂₈₀≦0.01 (at least 5 column volumes). Wash the column with BufferC [8M urea, 0.1M NaH₂PO₄, 0.01 M Tris-HCl, pH 6.3] until the OD₂₈₀≦0.01.The protein is eluted with Buffer C plus 0.25 mM Imidazole, collectingthirty 1 ml fractions.

Record the OD₂₈₀ of each fraction. Pool aliquots of the purified proteininto dialysis tubing (Spectra/Por Cellulose Ester Membrane MW cutoff=8000; Spectrum Medical Industries, 1100 Rankin Rd. Houston, Tex.77073-4716), and equilibrate in cold (4° C.) BufferC. Adjust theconcentration of the aliquots to ≦1 mg/ml using a standard commercialprotein quantitation kit (Bio-Rad Microassay, Bio-Rad Labs, 2000 AlfredNoble Dr., Hercules, Calif. 94547, 1-800-4BIORAD), with BSA diluted inBuffer C as the standard. Perform step dialysis of the protein in thecold (4° C.) beginning with Buffer C and reducing the molarity of theurea by whole number increments. Dialyze for one hour in each solution,ending in 1×PBS. Analyze the protein by (10%) SDS-PAGE running ˜2 μlprotein per well to verify protein size and quantity. The molecularweight of RIHisEae is 101 kDa.

Alternatively, add protein to dialysis tubing, dialyze straight into1×PBS. Quantitate the protein using a standard commercial proteinquantitation kit (Pierce BCA Protein Assay Kit, Pierce, P.O. Box 117,Rockford, Ill. 61105), aliquot, and store at −20° C. As with the firstalternative, analyze the protein by (10%) SDS-PAGE running ˜2 μl proteinper well to verify protein size and quantity.

Upon enrichment of his-tagged intimin, the material derived is analyzedfor level of purity by SDS-PAGE. A 10% SDS-PAGE gel is loaded with a 2μl sample of enriched his-tagged intimin and electrophoresed at 200 Vfor one hour. Molecular weight markers are included on the gel for sizecomparison. When the gel is stained with Colloidal Coomasie stain(Sigma, St. Louis, Mo.), the most prominent appears at ˜101 kDa. Severalother less prominent high molecular weights bands also appear. When thegel is stained with silver stain (BioRad, Richmond, Calif.) according tothe instructions of the manufacturer, very slight high molecular weightbands appear, as well as several more prominent bands at low molecularweights, the most prominent band appearing around 29 kDa. The enrichedproduct preferably contains approximately 70-80% of the full-length(i.e., 900 out of 935 predicted amino acids) intimin. Preferably theenriched product contains no more than 25% contaminants (i.e.,non-intimin related molecules), more preferably no more than 20%contaminants, still more preferably no more than 10% contaminants.

EXAMPLE III

Purification of Enriched Histidine-tagged Intimin

An enriched preparation of his-tagged intimin, generated as described inExample II above, is purified by techniques known to those skilled inthe art, including, but not limited to, high performance liquidchromatography (HPLC), gel column chromatography, and SDS-PAGE.

With the SDS-PAGE method, an enriched preparation of his-tagged intiminis separated on a 10% polyacrylamide gel and visualized, for example, bystaining an analytical lane with Colloidal Coomasie strain (Sigma, St.Louis, Mo.). The high molecular weight full-length intimin band can beexcised from the preparative gel with a razor, and stored at 4° C. priorto immunization. Less than full-length fragments of intimin, i.e.portions of intimin, and/or intimin conjugated to one or more antigenscan similarly be excised from the gel.

Regardless of the method used to purify intimin, or portion thereof, thepurified protein as used herein refers to a population of polypeptidesconsisting solely of intimin or portions or intimin, optionally taggedwith histidine. It has been recognized in the art that the population ofpolypeptides expressed from a fragment of DNA containing only one openreading frame encoding intimin (and intimin-like proteins) can separateinto multiple bands on an SDS-PAGE gel. McKee et al., Infection &Immunity, 64(6):2225-2233 (1996), Jerse et al., Proc. Natl. Acad. Sci.USA 87:7839-7843 (1990), and Isberg, Cell 50:769-778 (1987). Thus,purified intimin, as well as portions of intimin and intimin conjugatedwith one or more antigens, may be visualized as multiple bands on anSDS-PAGE gel.

EXAMPLE IV

A. Adherence Assay

Adherence of E. coli to either HEp-2 or HCT-8 cells is assessed by amodification of the method of Carvioto et al. Curr. Microbiol. 3: 95-99(1979). Specifically, overlay semiconfluent monolayers of HEp-2 cells onglass coverslips in 24 well tissue culture dishes or in 8 well PERMANOX™Chamber Slides (Nune, Naperville, Ill., chamber slides composed of anoxygen-permeable plastic polymer,) with adherence assay medium (EMEM, orEagle's Minimum Essential Medium supplemented with 0.4% sodiumbicarbonate and 1% mannose) which contain 20 μl/ml (v/v) of an overnightculture of the bacteria to be tested in LB broth.

Each inoculum contains ≧10⁷ bacteria (described below) which results inan approximate multiplicity of infection (MOI) of 100:1. The infectedmonolayers are incubated at 37° C. in a 5% CO₂ atmosphere. After threehours, the medium, which contains the nonadherent bacteria, is aspiratedand the monolayers washed once with sterile 10 mM phosphate bufferedsaline, pH 7.4 (PBS: sodium chloride, sodium phosphate dibasic, andpotassium phosphate monobasic).

Fresh adherence assay medium is added to the cells with adherentbacteria, and the infected cells are then incubated for an additional 3hours. The monolayers are then washed six times with PBS to removenonadherent bacteria. Each wash is gently removed by aspiration in anattempt to avoid disturbing the monolayers. Each assay is done ≧2 timesand duplicate slides are prepared to permit both Giemsa andFITC-phalloidin (FAS) staining to visualize binding and associatedsequelae.

For Giemsa staining, the HEp-2 cells and adherent bacteria are fixedwith 70% (v/v) methanol (glass coverslips) or graded acetone washes(chamber slides) and stained with 1:10 Giemsa (Sigma) for 20 minutes. Toassess the FAS phenotype, the FITC-Phalloidin (Sigma) staining procedureof Knutton et al. Infect. Immun. 57: 1290-1298 (1989) is used.Phalloidin is a mushroom phallotoxin that specifically bindsfilamentous, not globular, actin. FITC-phalloidin-stained preparationsare examined by both phase contrast and fluorescent microscopy using anOlympus model GHS microscope with a model BH2-RFL reflected lightfluorescence attachment (Olympus Optical Co., Ltd., Tokyo, Japan).

Adherence assays with HCT-8 cells are done by the procedure describedabove for HEp-2 cells, but the bacteria are allowed to interact with theHCT-8 cells for 2.5 hours before the first wash and an additional 2.5hours before terminating the assay. All assays with HCT-8 cells arecarried out in 8 well PERMANOX™.

B. Construction of a Bacteria for Use in the Assay: An EHEC eae mutant.

To create an in-frame deletion in the chromosomal copy of the eae genein a particular strain of EHEC, strain 86-24, the wild-type copy of thegene is replaced by double homologous recombination with aninternally-deleted copy of eae (FIG. 13). Plasmid pEB290 (FIG. 14)encloses most of the eae structural gene and is constructed from a PCRproduct amplified from the 86-24 chromosome with primer MM 1 (MM1=ATAACATGAGTACTCATGGTTG(SEQ ID NO:18); starts; at the second codon ofthe eae structural gene and includes a ScaI restriction site) incombination with primer MM2 (MM2=TCTAGAGAGAAAACGTGAATGTTGTCTCT(SEQ IDNO.2)).The resultant 2,953 base pair fragment derived by PCR is digestedwith the ScaI and XbaI and ligated into pBluescript SK⁺ (Stratagene)that is restricted with SmaI and XbaI. DNA sequencing of the ends of thepEB 290 insert reveals that the 3′ 250 base pairs are lost.

Plasmid pEB290 is transformed into E. coli strain GM119 [dam-6, dcm-3,[Arraj, J. A. and Marinus, M. G. J. Bacteriol. 153:562-565 (1983)] toobtain unmethylated DNA which is sensitive to the restrictionendonuclease BclI. Plasmid DNA is isolated (Maniatis, et al., Molecularcloning: a laboratory manual. Cold Spring Harbor (1982)) and restrictedwith Bc/I to remove an internal 1125 bp fragment from the gene. Theresulting sticky ends are ligated to each other to create pEB300 (FIG.15).

The deleted eae gene is excised by digesting pEB300 with XbaI andHindIII, and the fragment containing the eae sequence is ligated intothe BamHI site of the suicide vector, pAM450 (FIG. 16) to form pEB305.Plasmid pAM450 is a derivative of pMAK705 (Hamilton et al., J.Bacteriol., 171:4617-4622 (1989)) with three features. First, it has atemperature sensitive (ts) origin of replication. Second, the plasmidcarries the sacB/R locus from Bacillus subtilis, rendering the hoststrain sensitive to sucrose (Gay et al., J. Bacteriol 164:918-921(1985)); Lepesant et al., Marburg. Mol. Gen. Genet. 118:135-160 (1972)).Third, the plasmid encodes ampicillin resistance. These features allowhomologous recombination and positive selection for a secondrecombination event resulting in resolution and loss of vectorsequences. The insertion of the deleted eae gene (from pEB300) into thesuicide vector (pAM450) results in the plasmid called pEB305 (FIG. 17).

The suicide:eae construct, pEB305, is transformed into wild type EHECstrain 86-24 by electroporation (Sizemore, et al., Microb. Pathog.10:493-499 (1991)). Double recombinants that have been cured of thevector sequences are selected by growth on medium containing sucrose andthen screened for ampicillin sensitivity (Blomfield et al., Mol.Microbiol., 5:1447-1457 (1991)). Transformants that have been cured ofthe suicide vector sequences are sucrose resistant, ampicillinsensitive, and able to grow equally well at 30° and 42° C. Deletion ofthe chromosomal eae sequences is confirmed by: (i) the reduced size ofthe eae fragment after PCR amplification with primers MM1 and MM2; (ii)Southern blot analysis of the mutated chromosomal DNA; (iii) loss ofrestriction sites within the deleted region of the eae gene; and (iv)the inability of an internal probe to recognize the mutated chromosome.

The resulting in-frame deletion mutant of EHEC strain 86-24 strain isdesignated 86-24eaeΔ10. The mutation is confirmed to be in frame by invitro transcription and translation analysis of the PCR-derived productfrom 86-24eaeΔ10. A truncated protein product of the predicted size,about 68,000 Da, is identified by [³⁵S] methionine labeling of thetranslation product. The eae mutant strain is identical to wild type86-24 in all characteristics, including: growth in LB broth,agglutination with O157 and H7 antisera, inability to ferment sorbitol,and growth on MacConkey agar at 37° C.

Those of ordinary skill in the art will recognize that other methods ofcreating strains of EHEC that are mutated in eae and do not retainbinding ability are possible and may be substituted.

C. The Role of eae in EHEC Adherence in Vitro

The isogenic strains, 86-24, 86-24eaeΔ10 and 86-24eaeΔ10 carrying pEB310are tested for adherence to HEp-2 and HCT-8 cells. Wild type 86-24 formsmicrocolonies when the bacteria interact with HEp-2 or HCT-8 cells. M.L. McKee & A. D. O'Brien, Infection & Immunity 63:2070 (1995). Thislocalized adherence is FAS (fluorescence actin staining) positive whichindicates the polymerization of F-actin at the site of bacterialattachment (i.e., the expected result). The mutant 86-24eaeΔ10 is unableto adhere to HEp-2 cells. When eae is introduced into 86-24eaeΔ10 oneither pEB310 or pEB311, the LA/FAS (LA=localized adherence ormicrocolony formation) phenotype is fully restored, an observation whichdemonstrates that intimin alone complements the eae mutation. Since bothof the clones complement the eae mutant, the native promoter for eae ispresent in the PCR amplified sequences.

D. Effect of Adding Exogenous His-intimin Fusion Proteins

The adherence assay also may be used to evaluate the effect ofexogenously added His-intimin fusion proteins on the binding capabilityof 86-24eaeΔ10 and the binding capability of wild-type strain 86-24. Inthis case, the purified His-intimin fusion proteins are added to theepithelial cell monolayers before addition of bacteria as indicated ineach experiment.

HEp-2 cells are incubated with 20 ng-20 μg of RIHisEae for 30 minutesprior to the addition of 86-24 to the monolayer. The infected monolayersare then washed extensively, stained with FITC-phalloidin, and observedmicroscopically. The fusions enhance binding wild type strain of 86-24to HEp-2 cells. The size of the 86-24 microcolony as well as the totalnumber of HEp-2 cells with adherent microcolonies increases as theconcentration of RIHisEae increases. At high doses (20 μg), the fusionprotein causes the HEp-2 cells to show aberrant appendages andprocesses. For this reason, 1-2 μg is the most preferred dose forfurther studies.

When added exogenously to HEp-2 cells, RIHisEae complements theHEp-2-cell binding defect (or restores binding capability) of86-24eaeΔ10. The shorter fusion protein, RVHdHisEae, also complementsfor adherence. A similar amino terminal fusion of histidine residues tomouse dihydrofolate reductase (His-DHFR) does not enhance the adherenceof 86-24. Moreover, the plasmids that encode the intimin fusionproteins, pEB312 and pEB313, are able to complement 86-24eaeΔ10 forattachment in vitro. Thus, such studies indicate that the proteinsencoded by pEB312 and pEB313 are sufficient to confer adherence.

As noted above in Example I, the fusion proteins localize to theinsoluble pellet fraction after sonic disruption of the host strains,indicating that these proteins are localized to the membrane. PlasmidpQE16, which encodes the His-DHFR fusion, does not complement86-24eaeΔ10 (data not shown). That the irrelevant protein fusion withthe histidine residues does not confer HEp-2 cell adherence on the eaemutant indicates that the histidine residues added to intimin are notresponsible for the activity observed for the exogenously addedHis-intimin fusions. The enhancement or complementation of EHEC bindingto HEp-2 cells observable with exogenous RlHisEae and RVHdHisEaeindicates that intimin interacts with both the bacteria and theepithelial cell.

EXAMPLE V

The Role of eae in Vivo—Gnotobiotic Piglet Infection Model

The role of intimin in intestinal colonization, A/E lesion formation,and EHEC-mediated colitis and diarrhea in the gnotobiotic piglet isevaluated by the method of Francis, et al. (Francis et al.,Infect.Immun., 51:953-956 (1986)). Both pairs of piglets inoculated with thewild-type parent strain, 86-24 develop diarrhea and have edema in themesentery of the spiral colon at necropsy.

Histologically, strain 86-24 primarily colonizes the cecum and spiralcolon. Histologically and by culture, no evidence of bacterialdissemination to the liver, kidney, lung, or brain is detected. Intimatebacterial adherence and A/E lesions, as described by Staley (Staley etal., Vet. Pathol. 56:371-392 (1969)) and Moon (Moon et al., Infect.Immun., 41:1340-1351 (1983)) for EPEC, are evident by both light andelection microscopy (EM) examination of cecum and colon of pigletsinfected with 86-24. A/E lesions include the accumulation ofelectron-dense material at the site of attachment. In some areas,sloughed enterocyte fragments and microvilli with attached bacteria arenoted in the gut lumen. In histologic sections of the cecum and spiralcolon of piglets infected with 86-24, an inflammatory infiltrate isseen. Inflammation is characterized by scattered neutrophils in thelamina propria and mild diffuse accumulation of serous fluid andperivascular lymphocytes and macrophages in the submucosa.

Both piglets inoculated with the mutant strain, 86-24eaeΔ10 have formedfeces at necropsy. Histologically and by EM examination, there is noevidence that strain 86-24eaeΔ10 is able to colonize piglet intestineand cause the A/E lesion. The few bacteria seen by light and EMexamination are in the mucus overlying the mucosal epithelium of thececum and spiral colon. One of two piglets inoculated with 86-24eaeΔ10has slight mesocolonic edema, but no other gross or microscopic lesionsare seen in either piglet.

Piglets inoculated with 86-24eaeΔ10(pEB310) have pasty feces andmesocolonic edema at necropsy. Strain 86-24eaeΔ10 (pEB310) intimatelyadheres to mucosal enterocytes and causes A/E lesions in the cecum andspiral colon. Histologically, perivascular lymphohistiocytictyphlocolitis, similar to that caused by wild type 86-24 is also seen.

Similar experiments are conducted in a colostrum-deprived newborn calfmodel, showing that intimin is necessary to provoke A/E lesions in thegut as well as to evoke E. coli O157:H7 strain 86-24-mediated diarrhea.(A. D. O'Brien, M. R. Wachtel, M. L. McKee, H. W. Moon, B. T. Bosworth,C. Neal Stewart, Jr., and E. A. Dean-Nystrom. “Intimin: Candidate for anEscherichia coli O157:H7 Anti-Transmission Vaccine”. Abstract of the32nd Joint Conference on Cholera and Related Diarrheal Diseases,Nagasaki, Japan, Nov 14-16, 1996, the disclosure of which isincorporated herein by reference.) These experiments also demonstratethat by 2 days post-infection the numbers of infecting organisms in thelower bowel are significantly less in the animals fed the eae mutant ora non-pathogenic E. coli strain than in the calves fed the wild type orthe eae mutant with the complementing clone.

EXAMPLE VI

Recognition of EHEC Proteins by HC Patient Sera

Convalescent immune sera tested from hemorrhagic colitis patients(kindly provided by T. Barrett at the Centers for Disease Control andPrevention, Atlanta, Ga.) react with PT_(T7)-expressed intiminpreparations (i.e., his-intimin expressed by pEB310 and pEB311) in aWestern immunoblot. To decrease reactivity of the hemorrhagic colitispatients' sera with E. coli proteins in the expression system, serasamples are adsorbed with whole cell extracts of DH5α transformed withpGP1-2 and pBRKS⁻ (the expression vector). After adsorption, the normalsera controls recognize only proteins in the ammonium sulfateconcentrated fraction of the intimin preparations but no longer reactwith proteins expressed from pEB310 or the vector control. Afteradsorption, the HC patient sera still recognize many E. coli proteins,but the reaction with intimin remains strong.

EXAMPLE VII

Administration of His-Intimin to Patients

The following example provides the administration of his-intimin topatients in order to stimulate a protective immune response. Aprotective immune response is one that elicits sufficient antibody topermit a patient to avoid infection, decrease the significance orseverity of an infection, or decrease the ability of bacteria tocolonize the gastrointestinal tract.

Methods of administration of his-intimin include, but are not limitedto, injection (including, but not limited to, intraperitoneal,intravenous, subcutaneous, and intramuscular) of his-intimin directlyinto the patient to elicit an immune response, ingestion or by gavage ofhis-intimin alone or with food, and intra-nasal inoculation withhis-intimin, which promotes binding of intimin to receptors ofepithelial cells in the naso-pharynx.

When the his-intimin is ingested, the protein is contained within a gelcapsule, liposome, or attached to an inert substance to aid in passageof the inoculum through the stomach. As the fusion protein is acidstable, it also is ingested by itself or may be mixed into a foodproduct. A preferred method of administration is in a fusion protein, ofhis-intimin and SLT (Shiga-like toxin). A his-intimin-SLT fusion proteinis bound to SYNSORB (SynSorb Biotech, Inc., 1204 Kensington Rd, N.W.,Calgary, Alberta, Canada, T2N3P5), which has a receptor for SLT, via theSLT-receptor interaction. The SYNSORB construct is mixed with chocolatepudding and fed to children.

Purified RiHisEae (His-tagged Eae, 900/935 amino acids), as well as thethird third portion of intimin (encoded by pMW103), are stable afterincubation at pH 2.0 at 37° C. for 24 hr. This indicates that a His-Eaefusion can pass through the stomach unharmed or undegraded. Moreover, innature Eae is expressed on the outer membrane of the bacterium and itstill promotes intimate adherence after passing through the stomach,indicating its resistance to acidic environments.

Ingestion or intra-nasal inoculation stimulates local immunity, whichthwarts future colonization by EHEC and EPEC. Cross-immunity throughhomology is stimulated to Hafnia alvei and Citrobacter rodentium,Yersinia sp. and other bacterial species having intimin-like proteins.Although it is not necessary to quantitate the degree of cross-immunityconferred by administration of intimin in order to benefit from aprotective immune response to infection by bacteria other than EHEC thatexpress intimin-related proteins, an assay for such protection isdescribed in Example IX. The assay permits assessment of the efficacy ofintimin antibodies on blocking interaction with epithelial cells bypathogens known to have intimin-like binding proteins.

In another embodiment, injection of his-intimin into cow udders leads toan immune response in the cow. Antibodies against the protein arepresent in the cow's milk. Calves that drink the milk are passivelyimmunized until they can be actively immunized by the method of choice.Alternatively, his-intimin may be fed to cows or introduced into thecow's feed. The presence of his-intimin introduced in this way alsostimulates an antibody response in the cows so that antibodies areproduced and appear in the cows' milk.

Another embodiment involves the administration of nucleic acid vaccines.His-intimin is injected into a patient as naked eae DNA, or the DNA isdelivered to the body by a carrier system such as retroviruses,adenoviruses, or other carriers known in the art. Followingadministration, the patient mounts an immune response againsttransiently expressed foreign antigens.

Currently nucleic acid vaccines, in general, are nearing clinicaltrials. This approach to vaccines involves delivering the DNA encodingthe desired antigen into the host by inserting the gene into anonreplicating plasmid vector (Marwick, C. JAMA 273:1403 (1995);reviewed in Vogel, F. R. and N. Sarver. Clin. Microbiol. Rev. 8:406(1995)).

The first published demonstration of the. protective efficacy of such avaccine has shown that intramuscular injection of plasmid DNA encodinginfluenza A virus (A/PR/8/34) nucleoprotein (NP) elicited protectiveimmune responses in BALB/c mice against a heterologous strain ofinfluenza virus (A/HK/68) (Ulmer, J. B. et al. Science 259:1745 (1993)).Immunized animals had reduced virus titers in their lungs, decreasedweight loss, and increased survival compared with challenged controlmice. Both NP-specific cytotoxic T lymphocytes (CTL's) and NP antibodieswere generated. The NP antibodies were ineffective at conferringprotection, but the CTL's killed virus-infected cells and cells pulsedwith the appropriated major histocompatibility complex classI-restricted peptide epitope.

Another study has shown that intramuscular injection of plasmid DNAencoding influenza virus A/PR/8/34 hemagglutinin resulted in thegeneration of neutralizing antibodies that protected mice against aheterologous lethal influenza virus challenge (Montgomery, D. L. et al.DNA Cell Biol. 12:777 (1993)).

Practice of the invention by this method can be accomplished byreference to the aforementioned articles incorporated herein byreference, in particular Montgomery, D. L. et al. DNA Cell Biol. 12:777(1993). The eae locus is described in FIG. 5 according to restrictionsites and according to its sequence in FIG. 3, for strain CL8, and itssequence in strain 933, shown in FIG. 4.

EXAMPLE VIII

A. Conjugation of Antigens from Various Pathogens to His-intimin toElicit an Immune Response Against Both Eae and the Conjugated Antigen

Antigens (Ag) and haptens from various pathogens are conjugated to ahistidine-tagged intimin molecule. This fusion protein is used as aninoculum with intimin acting as the carrier to target binding tointestinal epithelial cells. This conjugate protein can be designed inany of the following configurations: N-His-intimin-Ag—C,N—Ag-intimin-His-C, N-His-Ag-intimin-C, N-intimin-Ag-His-C,N-intimin-His-Ag—C, or N-Ag-His-intimin-C.

The size of intimin varies with the size of the antigen that is to befused, and the number of antigens to which the intimin is fused as wouldbe recognized by those in the art. The variables to be considered in thedesign of such a fusion protein are: (1) foreign antigen; (2) size ofintimin used, which can be of whatever size that retains bindingfunction as described above; (3) fusion order N-C; and (4) method ofconjugation, such as genetic, as in cloning and expressing a fusionprotein, and chemical, although additional methods are readily apparentto those ordinarily skilled in the art. (D. V. Goeddel, “Systems forHeterologous Gene Expression,” Meth. Enzymol., Vol. 185, Academic Press,New York, 1990.; K. Itakura, “Expression in E. coli of a chemicallysynthesized gene for the hormone somatostatin,” Science, 198: 1056-1063(1977); and D. V. Goeddel et al., “Expression of chemically synthesizedgenes for human insulin,” Proc. Natl. Acad. Sci. USA, 281: 544-548(1979)).

Delivery of this coupled antigen occurs using the same mechanisms asthat of a histidine-tagged intimin alone, as set forth above in ExampleVII.

Haptens and antigens may derive from but are not limited to bacteria,rickettsiae, fungi, viruses, parasites, drugs, or chemicals. They mayinclude, for example, small molecules such as peptides,oligosaccharides, and toxins. Certain antimicrobial drugs,chemotherapeutic drugs having the capacity of being absorbed on themucosal surface may also be coupled to intimin. The antigens andpolysaccharides that may be coupled to intimin and administered tostimulate a protective immune response may include those shown below inTable 1.

TABLE 1 Antigens and/or polysaccharides from: Bordetella pertussisBorellia burgdorferi Campylobacter sp. including C. jejuni Candidaalbicans, other Candida Chlamydi trachomatis and pneumoniae (TWAR)Citrobacter rodentium Clostridium sp., including C. botulinum, C.difficile, C. perfringens, C. tetani, (including tetanus toxoid vaccine)Coronaviruses Corynebacterium diphtheriae, including diptheria toxoidvaccine Cryptococcus neoformans Entamoeba histolytica Escherichia colisp. including ETEC (enterotoxigenic E. coli), EAggEC (enteroaggregativeE. coli), EPEC (enteropathogenic E. coli), EHEC (enterohemmorhagic E.coli), EHEC SLT subunits or toxoid EIEC (enteroinvasive E. coli), UPEC(uropathogenic E. coli), including E. coli endotoxin, J5 antigen (LPS,Lipid A, Gentabiose), O polysaccharides (serotype specific) EHECHaemophilus influenza, including H. influenza type b (polyribosephosphate) Hafnia alvei Helicobacter pylon Hepatitis A, B, A, and othersHuman immunodeficiency virus I and II (GP120, GP41, GP160, p24, andothers) Histoplasma capsulatum Klebsiella species, includingpolysaccharides (serotype specific) Legionella species, including L.micdadei, L. pneumophila Listeria monocytogenes Mycobacterium species,including M. avium, M. kansasii, M. tuberculosis Mycoplasma Neisseriaspecies, including N. gonorrhoeae, N. meningitidis (including serotypespecific or protein antigens) Nocardia asteroides Plasmodium speciesPneumocystis carinii Polio virus Pseudomonas aeruginosa, includingserotype specific polysaccharides Rabies virus Rhinovirus RickettsiaRotavirus Salmonella sp., including S. cholerasuis, S. enteriditis, S.typhi, S. typhimurium Shigella species, including S. flexneri, S.sonnei, S. boydii, S. dysenteriae Staphylococcus sp., including S.aureus, polysaccharides from types 5 and 8 (serotype specific and commonprotective antigens), S. epidermidis, serotype polysaccharide I, II, andIII (and common protective antigens) Streptococcus species, allserotypes including S. pneumoniae (all serotypes), S. pyogenes,including group A, group B (serotypes Ia, Ib, II, and III) Treponemapallidum Varicella zoster Vibrio cholerae Yersinia species, including Y.pestis, Y. pseudotuberculosis, Y. enterocolitica

The sizes of his-intimin that may be conjugated to antigens appearing inTable 1 include RlHisEae (900/935 aa, EcoRI-HindIII fragment of pEB313)and RVHindHis (604/935 aa, EcoRV-HindIII fragment of pEB313), as setforth above in Example I. Those of ordinary skill in the art willrecognize that additional fragments of varying lengths having adherenceactivity may be selected within the spirit and scope of the invention.The efficacy of the fragments considered for selection may be assessedaccording to the procedures described in Example IV.

B. Construction of a Plasmid Expressing N-His-IcsA-Intimin-C

Shigella flexneri causes bacillary dysentery in humans by invadingepithelial cells of the colonic mucosa (Labrec et al. J. Becteriol.88:1503-1518, (1964)). A. 120 kDa outer membrane protein, called IcsA,is necessary for intra- and intercellular spread of this organism(Bernardini et al. Proc. Natl. Acad. Sci. USA.86:3867-3871, (1989); Lettet al. J. Bacteriol. 171:353-359, (1989)). An iscA mutant (SC560) wasreasonably well tolerated by orally infected macaque monkeys andelicited protection against homologous challenge (Sansonefti et al.Vaccine 9:416-422, 1991).

The following protocol may be used (FIG. 18):

Transform pEB313 into a dam⁻ host, such as DM1 (Gibco BRL, P.O. Box 68,Grand Island, N.Y. 14072, 1-800-828-6686). Digest pEB313/Clal/HindIII,isolate 1796 bp fragment (this fragment encodes the last 547 amino acidsof intimin). Ligate into pBluescriptSK+/ClaI/HindIII(pBluescriptSK+available from Stratagene, 11011 N. Torrey Pines Rd., LaJolla, Calif. 92037, 1-800-424-5444). Call this plasmid pEae1. DigestpHS3192 with AvaI, fill in the end with Klenow fragment, digest withClal, isolate 2490 bp fragment [this fragment encodes 2923 bp or 974aa's from base pair #706-3629; the ORF of icsA spans from bp#574-3880,this is 3306 bp and encodes 1102 aa's; reference for sequence of icsA isLeft et al., J. Bacteriol. 171:353 (1989)](pHS3192 available from P.Sansonetti (ref Bernardini, M. L. et al. Proc. Natl. Acad. Sci. USA.86:3876 (1989)). Ligate the 2490 bp fragment into pEae1 digested withClal and HincII, producing a plasmid called pEae2. Using theserestriction enzymes, the reading frames of icsA and eae remain in frame.Digest pEae2 with XhoI and HindIII, isolate the 4286 bp fragment; ligateinto pQE 32 (QIAGEN) digested with SmaI and HindIII. This ligation willmaintain the proper reading frame of both genes with the promoter. Theresulting plasmid is called plcsA-Eae.

Alternatively, one could fuse two genes:in frame by cloning with PCR,followed by ligation into the appropriate pQE vector. This technique iswell known to those of ordinary skill in the art.

C. Preparation of a Conjugate Vaccine Using His-intimin as the ProteinCarrier

While any polysaccharide could be used, in this vaccine the capsular Vipolysaccharide of Salmonella typhi is used. Purify His-intimin as inExample II; this would be conjugated to Vi (purified from S. typhiaccording to established procedures (Szu et al. J. Exp. Med. 166:1510(1987)). The conjugation will proceed using standardprotein-polysaccharide conjugation technology well known to those in theart. Methods of conjugation are well known to those of ordinary skill inthe art, and include the heteroligation techniques of Brunswick, M. etal., J. Immunol. 140:3364, 1988. See also Chemistry of Proteinconjugates and Crosslinking CRC Press, Boston (1991).

Techniques to conjugate moieties to primary or secondary carriers arewell known to those skilled in the art, and include, in part, couplingthrough available functional groups (such as amino, carboxyl, thio andaldehyde groups). See S. S. Wong, Chemistry of Protein Conjugate andCrosslinking CRC Press (1991); and Brenkeley et al. Brief Survey ofMethods for Preparing Protein Conjugates With Dyes, Haptens andCross-linking Agents, Bioconjugate Chemistry 3 #1 (January 1992).

A vaccine such as that described in this example would provide aprevention of diarrheal pathogens to include both those organisms thatexpress intimin (or intimin-like proteins), as well a diarrheal pathogenthat expresses Vi.

Any combination of intimin plus other antigens from other diarrhealpathogens can be combined. In addition, if polysaccharides were usedfrom organisms that produce other diseases, such as pneumococcalpolysaccharides, the intimin-polysaccharide vaccine would be useful forprevention of multiple diseases. Delivery of a vaccine againstrespiratory pathogens will preferentially be done directly to therespiratory tract; ingested pathogens through ingestion.

EXAMPLE IX

Generation and Testing of Adherence-blocking Anti-intimin Antibodies:Polyclonal and Monoclonal

High titer polyclonal anti-intimin antisera are elicited uponintraperitoneal injection of RlHisEae into mice, rabbits, and goats.Testing of antibody titer and an antibody effectiveness assay are shown.The generation of monoclonal antibodies is also described.

A. Generation of Polyclonal Antibodies

Various techiques can be used to prepare antibodies against full-lengthintimin or various portions thereof in various animals. Several of thesetechniques are described below. As would be recognized by one skilled inthe are, polyclonal antibodies can be generated from intimin andportions of intimin that are not his-tagged and from intimin-likeproteins and portions thereof.

1. Generation of Mouse Anti-RIHisEae Polyclonal Antibodies

The technique of Harlow, E. and D. Lane (eds) Antibodies—a LaboratoryManual. Cold Spring Harbor, New York (1988) may be followed. The generalprocedure is outlined herein. Take pre-bleeds of each mouse to beimmunized: Bleed from the tail vein into an eppendorf tube. Incubate at37° C. for 30 min, stir gently with a sterile toothpick (to loosen theclot), store overnight at 4° C. In the morning, spin 10 min/10,000 rpmin the microfuge, and collect the serum (i.e., supernatant; red bloodcells are the pellet). Store the serum at −20° C. The sera obtained willbe used as a negative control after the mice are immunized.

Inject a BALB/c mouse intraperitoneally with 25 μg of RIHisEae (usingTITERMAX®, an adjuvant such as Freund's Complete; adjuvant, according tothe instructions of the manufacturer (CytRx Corp., 154 Technology Pkwy.,Norcross, Ga. 30092, 800-345-2987). Wait 2 weeks, boost with anidentical shot, wait 7 days and bleed from the tail vein into aneppendorf tube. Incubate at 37° C. for 30 min, stir gently with asterile toothpick (to loosen the clot), store overnight at 4° C. In themorning, spin 10 min/10,000 rpm in the microfuge, and collect the serum.Store the sera at −20° C.

2. Generation of Mouse Anti-third Third Portion of Intimin PolyclonalSera

Mice are prebled by the tail vein as described above in Example IX, partA. The third third portion of intimin is enriched and dialyzed asdescribed above in Example II. Mice are injected with the third thirdportion of intimin mixed with TITERMAX® adjuvant, as described inExample IX, part A. After 3 boosts, mice are bled via the retro-orbitalsinus, and sera prepared in described Example IX, part A. Sera is testedby Western blot analysis, as described for the goat polyclonal sera insection 4 below. Sera is assayed for the capacity to block EHECadherence to HEp-2 cells as described above in Example IX, part C.

3. Generation of Rabbit Polyclonal Anti-Intimin Antibodies

Rabbit polyclonal sera is generated against the (1) first third, (2)second third, and (3) third third portions of intimin. Each specificsera is separately assayed in HEp-2 adherence assays for the capacity toblock adherence of EHEC to HEp-2 cells.

Preparation of First Third Portion of Intimin for Rabbit Immunization

Clone pMW101 is transformed into strain DH5αF'lacl^(Q). Induction ofprotein expression and purification of the His-tagged intimin fragmentover the Ni-NTA affinity resin is performed as described in the Qiagenmanual that accompanies their QIAEXPRESSIONIST® Ni-NTA resinpurification kit (Qiagen Inc., Chatsworth, Calif.). Eluted fractions aremonitored for protein content by A₂₈₀ and by Bradford analysis, using adye reagent from Bio-Rad (Hercules, Calif.). Peak fractions eluted fromthe Ni-NTA column (with 250 mM imidazole) are electrophoresed on 15%polyacrylamide gels with SDS for analysis and purification. To visualizethe proteins on the gels for analysis, both silver (Bio-Rad) andCoomassie Blue G-250 (Sigma) staining are used.

To purify the protein for immunization of animals to obtain antisera,the peak column fractions are run on preparative SDS polyacrylamidegels, and proteins are visualized with Copper stain (Bio-Rad). The bandof protein corresponding to the intimin fragment is excised from the gelwith a clean razor blade, and the gel slice is destained according tothe instructions provided with the Copper stain reagent. Protein is theneluted from the gel slice using an Electro-Eluter Model 422 (Bio-Rad)according to the manufacturer's instructions. The protein is thenconcentrated using a CENTRICON® 10 concentrator, an ultrafiltrationmicroconcentration membrane system with a 10 kilodalton molecular weightcut-off, from Amicon (Beverly, Mass.). The majority of the SDS in theeluted protein sample is removed by one of two methods. The first methodinvolves addition of phosphate-buffered saline (PBS) to the proteinsample, which causes precipitation of SDS. The majority of the proteindoes not precipitate, and the precipitate is not analyzed to determinewhat ions may also have precipitated. The SDS is pelleted bycentrifugation; and the supernatant, which contains most of the proteinand possibly some residual SDS, is removed and concentrated using aCENTRICON® 10 concentrator from Amicon (Beverly, Mass.).

The second method for removal of SDS involves preparing a column ofEXTRACTI-GEL® D Detergent Removing Gel, purchased from Pierce (Rockford,Ill.). The EXTRACTI-GEL® D Detergent Removing Gel is used according tothe instructions of the manufacturer. The purified protein isconcentrated as described above. Protein concentrations are determinedby Bradford analysis using dye reagent purchased from Bio-Rad and alsoby running different volumes of purified protein on a gel adjacent toaliquots of varying amounts of the original column fractions to comparethe amounts of proteins visually. Fractions of this purified protein areanalyzed by SDS-PAGE using both silver and Coomassie staining.

Preparation of Second Third Portion of Intimin for Rabbit Immunization

Purification of the His-tagged middle third fragment of the intiminprotein expressed from clone pMW102 is performed with the same methodsused for the N-terminal third, with the following instructions. SDS-AGEanalysis is done using 12.5% acrylamide gels. For gel-purification ofthe protein and electrolution, most of the preparative gels are stainedwith Copper stain as above; and one gel was stained with Coomassiebrilliant blue dissolved in water as described in Harlow, E. and D. Lane(eds.) Antibodies—a Laboratory Manual. Cold Spring Harbor, N.Y. (1988).Much of the SDS in the electroeluted protein fractions is precipitatedout by addition of PBS buffer. For concentration of the protein, AmiconCENTRICON® 30 concentrators, ultrafiltration microconcentration membranesystems with a 30 kilodalton molecular weight cut-off, are used(Amicon).

Preparation of Third Portion of Intimin for Rabbit Immunization

The third third intimin protein is enriched and dialyzed as describedabove in Example II. One mg of protein is run by SDS-PAGE on four(Small-Scale electrophoresis apparati) Protein in negatively stainedwith copper stain (BioRad, cat #161-0470, Richmond, Calif.) according tothe instructions of the manufacturer as follows: the gel is rinsed indH₂O for 45 seconds, stained in 1X copper stain for 5 minutes, andrinsed in dH₂O for 3 minutes. The gel is visualized against a blackbackground, and the ˜37 kDa protein band is cut from the gel with arazor. Purified gel slices are then de-stained in buffer (25 mM Trisbase, 192 mM glycine, 3X/10 min), wrapped in plastic wrap and stored at−20° C. prior to immunization.

Immunization of Rabbits

New Zealand white female rabbits (5 to 6 lbs) are immunized separatelywith the antigenes prepared as described above according to a schedulethat could be readily determined by one skilled in the art. An exampleof such a schedule is as follows:

DAY PROCEDURE 0 Prebleed/initial Inoculation, 100 μg Ag mixed withcomplete Freund's adjuvant 14 Boost, 50 μg mixed with incompleteFreund's adjuvant 21 Boost, 50 μg mixed with incomplete Freund'sadjuvant 35 Test Bleed 45 Boost, 50 μg mixed with incomplete Freund'sadjuvant 56 Test Bleed

The route of injection can be subcutaneous and/or intermuscular atmultiple sites. Sera derived from test bleeds is tested for specificrecognition of the antigen by Western Blot analysis, as described forthe goat polyclonal sera in section 4 below. When high titer recognitionof the antigen is achieved, as recognizable by one skilled in the art,the rabbit is exsanguinated to recover the antibodies. The large volumesample of blood is verified for specific recognition of the antigen byWestem Blot analysis.

Affinity Purification of Rabbit Anti-intimin Polyclonal Sera by WesternBlot

Rabbit anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin or intimin-likeproteins from the sera. (Harlowe, E. and D. Lane (eds) Antibodies—aLaboratory Manual. Cold Spring Harbor, N.Y. (1988), p. 498 or S. H.Lillie and S. S. Brown. Yeast. 3:63 (1987)). RIHisEae (0.250 mg) iselectrophoresed by SDS-PAGE (size: BioRad MiniProtean II minigel,BioRad, Richmond, Calif.), transferred to nitrocelullose, and stainedwith Ponceau S (Sigma, St. Louis, Mo.). A strip of nitrocellulosecontaining the full length His-intimin band (about 100 kDa) is excisedwith a razor, and the nitrocellulose strip containing the protein isincubated overnight at 4° C. in 2% milk/TBS-0.2% TWEEN®, a detergent,(ICI Americas, Inc., Wilmington, Del.), shaking gently. Thenitrocellulose strip is washed briefly in TWEEN®, and placed in acontainer on top of a piece of PARAFILM®, a moisture proof film sealant.(American National Can, Greenwich, Conn.). Rabbit sera is pipetted ontothe mini-Western blot (as much volume as will fit, about 400-500 μl),and wet paper towels are placed over the containing, not touching thenitrocellulose strip, followed by plastic wrap. The blot is shakengently for 5 hours, after which the sera (now called “depleted sera”) isremoved and saved for analysis. The strip is washed 3 times in PBS for10 minutes, and glycine buffer (150 mM NaCl, pH 2.3-with HCl) is added(as much volume as will fit onto the strip) for 30 minutes. Affinitypurified antibodies are pipetted off, and 1/10 volume Tris-HCl, pH 8.0is added. Antibodies so recovered are then neutralized with 1N NaOH andtested by Western blot analysis as described below.

Affinity Purification of Rabbit Anti-intimin Polyclonal Sera by AntigenAffinity Column

Rabbit anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin from the sera.Antisera raised against intimin or various portions thereof is purifiedusing an antigen affinity column using techniques known to those skilledin the art, such as those described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, N.Y. (1988).

The antigens (intimin or portions of intimin) are enriched as describedabove in Example II. Antigens may be further purified by electrophoresison an acrylamide gel followed by electroelution from a gel slicecontaining the protein as described below in part 4. Other methods maybe substituted for gel-purification and electroelution to further purifythe protein after elution from the Ni-NTA resin. These methods mayinclude, but are not limited to, ion-exchange column chromatography andgel filtration chromatography. After purification, the intimin proteinmay need to be dialyzed into an appropriate buffer for coupling toactivated beads to form the affinity resin for antisera purification.

Activated beads appropriate for coupling to the antigen are selectedbased on several properties: coupling reagent, binding group or matrix,ligand attachment, and stability of the final matrix (as listed inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988)). For example, the purified initimin (orportion of intimin) protein antigen is coupled to Affigel beads(Bio-Rad, Richmond, Calif.) according to the instructions of themanufacturer. A column of the activated beads coupled to the antigen isprepared and washed according to instructions of the manufacturer of thebeads. The column is then washed according to the method described inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988).

Ammonium sulfate precipitation is used to partially purify the sera inpreparation for the affinity column. Ammonium sulfate precipitation,resuspension of the protein pellet in PBS, dialysis of the solutionversus PBS, and centrifugation to clarify the solution are performed asdescribed in Harlow, E. and D. Lane (eds.) Antibodies—a LaboratoryManual. Cold Spring Harbor, N.Y. (1988).

Antisera that has been partially purified by ammonium sulfateprecipitation and dialysis versus PBS is passed over the antigenaffinity column as described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, N.Y. (1988). Theantisera may be passed over the column multiple times, as this may leadto more complete binding of antibodies to the column. The column is thenwashed and the affinity-purified antibodies are eluted and dialyzedagainst PBS as described in Harlow, E. and D. Lane (eds.) Antibodies—aLaboratory Manual. Cold Spring Harbor, N.Y. (1988).

Adherence Assays

Affinitiy purified polyclonal sera is assayed in HEp-2 cell adherenceassays for the capacity to block bacterial binding to HEp-2 cells usingthe method described below in Example IX, part C.

4. Generation of Goat Anti-RIHisEae Polyclonal Antibodies

Pre-bleeds are taken of potential goats to be immunized. Blood iscollected from the jugular vein with indirect vacuum. Sera is separatedfrom the whole blood, as described above in Example IX, section A, andtested by ELISA using RIHisEae as the adsorbent (as described in ExampleIX, section B, below for the ELISA and Example II above for theenrichment of RIHisEae), or by Western blot analysis as described below.The goat chosen for immunization has pre-immune sera with both (a) thelowest recognition of intimin by Western blot analysis and (b) thelowest titer against intimin by ELISA, and does not have the habit ofjumping out of the pasture.

Western Blot Analysis of Goat Anti-RIHisEae Polyclonal Sera

a. Generation of Whole Cell Lysates

Desired strains (for example: 86-24, 86-24eaeΔ10, DH5α, M15 pREP4pEB313) are grown overnight in LB containing the appropriate antibioticsat 37° C., with shaking. Cells (4.5 ml) are pelleted in an eppendorftube, and 500 μl sonication buffer (50 mM Na-phosphate pH 7.8, 300 mMNaCl) are added. Cells are sonicated in 15 second pulses on ice,aliquoted and frozen at −20° C.

b. Western Blot Analysis

Whole cell lysates generated as described above (2-5 μl) or purifiedRIHisEae (2 μl) are run by SDS-PAGE, transferred to nitrocellulose, andused for Western blot analysis of goat sera. The sera (primary antibody)is typically diluted 1:500 or 1:1000 for this purpose. The secondaryantibody used is swine anti-goat IgG conjugated to horseradishperoxidase (Boehringer Mannheim, Indianapolis, Ind.), diluted 1:2000.Pre-bleeds of goat sera usually contain several cross-reactive bandsthat are removed later by affinity purification.

Preparation of Purified RIHisEae (Antigen) for Immunization Into Goat

One mg of RIHisEae, generated as described in Example II above, is runby preparative SDS-PAGE. A small analytical lane is stained withColloidal Coomasie stain (Sigma, St. Louis, Mo.) and used for comparisonto the rest of the preparative gel. The high molecular weightfull-length intimin band (not stained, running at about 100 kDa) isexcised from the preparative gel with a razor, and stored at 4° C. priorto immunization.

Immunization of Goats with Antigen

Female goats (approximately one and a half years old, purebred Saanan orSaanan X LaMANCHA) are immunized separately with the antigens preparedas described above according to a schedule that could be readilydetermined by one skilled in the art. For example, the goat is given aprimary immunization of 500 μg of prepared RIHisEae mixed with CompleteFreunds adjuvant. At two week intervals the goat is boosted with 250 μgAg mixed with incomplete Freunds adjuvant. Test bleeds are begun afterthe goat has been immunized for a month, and continue until a highanti-intimin titer is reached, as defined by Western blot analysis,described above. When the sera recognizes intimin by Western blot, largeblood samples are taken (500 mls, resulting in about 250 mls sera) persession, with two week intervals between large bleeds. Resultinglarge-volume sera samples are verified for recognition of intimin byWestern blot analysis, as described above.

Affinity Purification of Goat Anti-intimin Polyclonal Sera by WesternBlot

Goat anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin from the sera.(Harlowe, E. and D. Lane (eds) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988), p. 498 or S. H. Lillie and S. S. Brown.Yeast. 3:63 (1987)). RIHisEae (0.250 mg) is electrophoresed by SDS-PAGE(size: BioRad MiniProtean II minigel, BioRad, Richmond, Calif.),transferred to nitrocelullose, and stained with Ponceau S (Sigma, St.Louis, Mo.). A strip of nitrocellulose containing the full lengthHis-intimin band (about 100 kDa) is excised with a razor, and thenitrocellulose strip containing the protein is incubated overnight at 4°C. in 2% milk/TBS-0.2% TWEEN® (ICI Americas, Inc., Wilmington, Del.);shaking gently. The nitrocellulose strip is washed briefly in TWEEN®,and placed in a container on top of a piece of PARAFILM (AmericanNational Can, Greenwich, Conn.). Goat sera is pipetted onto themini-Western blot (as much volume as will fit, about 400-500 μl), andwet paper towels are placed over the containing, not touching thenitrocellulose strip, followed by plastic wrap. The blot is shakengently for 5 hours, after which the sera (now called “depleted sera”) isremoved and saved for analysis. The strip is washed 3 times in PBS for10 minutes, and glycine buffer (150 mM NaCl, pH 2.3-with Hcl) is added(as much volume as will fit onto the strip) for 30 minutes. Affinitypurified antibodies are pipetted off, and 1/10 volume Tris-HCl, pH 8.0is added. Antibodies are then neutralized with 1N NaOH and tested byWestern blot analysis as described above.

Affinity Purification of Rabbit Anti-intimin Polyclonal Sera by AntigenAffinity Column

Rabbit anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin from the sera.Antisera raised against intimin or various portions thereof is purifiedusing an antigen affinity column using techniques known to those skilledin the art, such as those described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, N.Y. (1988).

The antigens (intimin or portions of intimin) are enriched as describedabove in Example II. Antigens may be further purified by electrophoresison an acrylamide gel followed by electroelution from a gel slicecontaining the protein as described below in part 4. Other methods maybe substituted for gel-purification and electroelution to further purifythe protein after elution from the Ni-NTA resin. These methods mayinclude, but are not limited to, ion-exchange column chromatography andgel filtration chromatography. After purification, the intimin proteinmay need to be dialyzed into an appropriate buffer for coupling toactivated beads to form the affinity resin for antisera purification.

Activated beads appropriate for coupling to the antigen are selectedbased on several properties: coupling reagent, binding group or matrix,ligand attachment, and stability of the final matrix (as listed inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988)). For example, the purified initimin (orportion of intimin) protein antigen is coupled to Affigel beads(Bio-Rad, Richmond, Calif.) according to the instructions of themanufacturer. A column of the activated beads coupled to the antigen isprepared and washed according to instructions of the manufacturer of thebeads. The column is then washed according to the method described inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988).

Ammonium sulfate precipitation is used to partially purify the sera inpreparation for the affinity column. Ammonium sulfate precipitation,resuspension of the protein pellet in PBS, and dialysis of the solutionversus PBS and centrifugation to clarify the solution is performed asdescribed in Harlow, E. and D. Lane (eds.) Antibodies—a LaboratoryManual. Cold Spring Harbor, N.Y. (1988).

The antisera that has been partially purified by ammonium sulfateprecipitation and dialysis versus PBS is passed over the antigenaffinity column as described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, New York (1988). Theantisera may be passed over the column multiple times, as this may leadto more complete binding of antibodies to the column. The column is thenwashed and the affinity-purified antibodies are eluted and dialyzedagainst PBS as described in Harlow, E. and D. Lane (eds.) Antibodies—aLaboratory Manual. Cold Spring Harbor, N.Y. (1988).

Adherence Assays

Affinitiy purified polyclonal sera is assayed in HEp-2 cell adherenceassays for the capacity to block bacterial binding to HEp-2 cells usingthe method described below in Example IX, part C.

B. ELISA to Test Titer of Antibodies

The technique of Harlow, E. and D. Lane (eds) Antibodies—a LaboratoryManual. Cold Spring Harbor, N.Y. (1988) may be followed. The generalprocedure is outlined below:

(1) bind RIHisEae to plastic microtiter plates at 50 ng/well in PBS.Incubate 2h/RT (room temp) or overnight at 40° C.

(2) wash plate 2×with PBS.

(3) block wells with 100 μl blocking solution [3% bovine serum albumin(Sigma Chemical, St. Louis, Mo.), 0.02% sodium azide (Sigma) inPBS—store stock at 4° C.] for 1-2 h at RT.

(4) wash plate 2×with PBS.

(5) primary Ab=50 μl test sera diluted in blocking solution for example,start with 1:50 and do eleven 1:2 dilutions, or start with 1:50 and doeleven 1:10 dilutions), incubate 2 h/RT.

(6) wash 4×with PBS.

(7) secondary Ab=goat horseradish-conjugated anti-mouse Ig, affinitypurified (Boehringer Mannheim Corp., 9115 Hague Rd., P.O. Box 50414,Indianapolis, Ind. 46250, 800-262-1640). Add secondary Ab diluted 1:500in blocking solution without azide. Incubate 1 h/RT.

(8) wash 4×with PBS.

(9) add 100 μl TMB Peroxidase substrate to each well (prepared accordingto the instructions of the manufacturer, BioRad Labs, 3300 RegattaBlvd., Richmond, Calif. 94804). Allow blue color to develop (no morethan 10 min). Stop the reaction with 100 μl H₂SO₄. Read the plate at 450nm.

A titer is defined as an absorbance value ≧0.2 units above that obtainedfor mouse pre-immune sera.

Anti-intimin antibodies may be administered to provide passive immuneprotection to a patient in need thereof. Moreover, anti-intiminantibodies obtained from animals may be used clinically in humans. Insuch cases, it is preferable to humanize the antibody. Such techniquesare well known to those of ordinary skill in the art. G. Winter et al.,“Man-made antibodies,” Nature, 349: 293-299 (1991); P. T. Jones et al.,“Replacing the complementarity-determining regions in a human antibodywith those from a mouse,” Nature, 321: 522-525 (1986); P. Carter et al.,“Humanization of an anti-p185^(HER2) antibody for human cancer therapy,”Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992). Such antibodies may begiven to the sibling of an infected patient to reduce the risk ofinfection of the sibling.

C. Western Blot Analysis of Anti-RIHisEae Polyclonal Sera

Polyclonal sera is assayed by Western blot analysis to verifyrecognition of intimin.

1. Generation of Whole Cell Lysates

Desired strains (for example: 86-24, 86-24eaeΔ10, DH5α, M15 pREP4pEB313) are grown overnight in LB containing the appropriate antibioticsat 37° C., with shaking. Cells (4.5 ml) are pelleted in an eppendorftube, and 500 μl sonication buffer (50 mM Na-phosphate pH 7.8, 300 mMNaCl) are added. Cells are sonicated in 15 second pulses on ice,aliquoted and frozen at −20° C.

2. Western Blot Analysis

Whole cell lysates generated as described above (2-5 μl) or purifiedRIHisEae (2 μl) are run by SDS-PAGE, transferred to nitrocellulose, andused for Western blot analysis of sera. The sera (primary antibody) istypically diluted 1:500 or 1:1000 for this purpose. The secondaryantibody is specific for the animal that is the source of the primaryantibody and is conjugated to horseradish peroxidase. Pre-bleeds of seramay contain several cross-reactive bands that are removed later byaffinity purification.

D. Affinity Purification of Anti-intimin Polyclonal Sera by Western Blot

Anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin from the sera.(Harlowe, E. and D. Lane (eds) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988), p. 498 or S. H. Lillie and S. S. Brown.Yeast. 3:63 (1987)). RIHisEae (0.250 mg) is electrophoresed by SDS-PAGE(size: BioRad MiniProtean II minigel, BioRad, Richmond, Calif.),transferred to nitrocelullose, and stained with Ponceau S (Sigma, St.Louis, Mo.). A strip of nitrocellulose containing the full lengthHis-intimin band (about 100 kDa) is excised with a razor, and thenitrocellulose strip containing the protein is incubated overnight at 4°C. in 2% milk/TBS-0.2% TWEEN® (ICI Americas, Inc., Wilmington, Del.);shaking gently. The nitrocellulose strip is washed briefly in TWEEN®,and placed in a container on top of a piece of PARAFILM® (AmericanNational Can, Greenwich, Conn.). Sera is pipetted onto the mini-Westernblot (as much volume as will fit, about 400-500 μl), and wet papertowels are placed over the containing, not touching the nitrocellulosestrip, followed by plastic wrap. The blot is shaken gently for 5 hours,after which the sera (now called “depleted sera”) is removed and savedfor analysis. The strip is washed 3 times in PBS for 10 minutes, andglycine buffer (150 mM NaCl, pH 2.3-with Hcl) is added (as much volumeas will fit onto the strip) for 30 minutes. Affinity purified antibodiesare pipetted off, and 1/10 volume Tris-HCl, pH 8.0 is added. Antibodiesare then neutralized with 1N NaOH and tested by Western blot analysis asdescribed above.

E. Affinity Purification of Anti-intimin Polyclonal Sera by AntigenAffinity Column

Rabbit anti-intimin polyclonal sera is affinity purified to removecross-reacting antibodies not specific for intimin from the sera.Antisera raised against intimin or various portions thereof is purifiedusing an antigen affinity column using techniques known to those skilledin the art, such as those described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, N.Y. (1988).

The antigens (intimin or portions of intimin) are enriched as describedabove in Example II. Antigens may be further purified by electrophoresison an acrylamide gel followed by electroelution from a gel slicecontaining the protein as described below in part 4. Other methods maybe substituted for gel-purification and electroelution to further purifythe protein after elution from the Ni-NTA resin. These methods mayinclude, but are not limited to, ion-exchange column chromatography andgel filtration chromatography. After purification, the intimin proteinmay need to be dialyzed into an appropriate buffer for coupling toactivated beads to form the affinity resin for antisera purification.

Activated beads appropriate for coupling to the antigen are selectedbased on several properties: coupling reagent, binding group or matrix,ligand attachment, and stability of the final matrix (as listed inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988)). For example, the purified initimin (orportion of intimin) protein antigen is coupled to Affigel beads(Bio-Rad, Richmond, Calif.) according to the instructions of themanufacturer. A column of the activated beads coupled to the antigen isprepared and washed according to instructions of the manufacturer of thebeads. The column is then washed according to the method described inHarlow, E. and D. Lane (eds.) Antibodies—a Laboratory Manual. ColdSpring Harbor, N.Y. (1988).

Ammonium sulfate precipitation is used to partially purify the sera inpreparation for the affinity column. Ammonium sulfate precipitation,resuspension of the protein pellet in PBS, and dialysis of the solutionversus PBS and centrifugation to clarify the solution is performed asdescribed in Harlow, E. and D. Lane (eds.) Antibodies—a LaboratoryManual. Cold Spring Harbor, N.Y. (1988).

The antisera that has been partially purified by ammonium sulfateprecipitation and dialysis versus PBS is passed over the antigenaffinity column as described in Harlow, E. and D. Lane (eds.)Antibodies—a Laboratory Manual. Cold Spring Harbor, New York (1988). Theantisera may be passed over the column multiple times, as this may leadto more complete binding of antibodies to the column. The column is thenwashed and the affinity-purified antibodies are eluted and dialyzedagainst PBS as described in Harlow, E. and D. Lane (eds.) Antibodies—aLaboratory Manual. Cold Spring Harbor, N.Y. (1988).

F. Assay for Blocking of Bacterial Binding by Antibodies to Intimin

To assess the effect of anti-intimin antibodies on EHEC adherence,mouse, rabbit, or goat anti-intimin antisera (or normal sera ascontrols) are added to EHEC bacteria suspended in adherence media, andthe bacteria-antisera mixtures are incubated at 37° C. for thirtyminutes prior to infection of HEp-2 cells. Antisera are maintained inthe adherence media throughout the assay. Adherence and related sequelaeare microscopically observed using GIEMSA and FITC-phalloidin (FAS)staining as described above.

To assess the effect of anti-intimin antibodies on adherence of otherbacteria having intimin-like proteins, mouse, rabbit, or goatanti-intimin antisera (or normal sera as controls) are added to EHECbacteria suspended in adherence media, and the bacteria-antiseramixtures are incubated at 37° C. for thirty minutes prior to infectionof HEp-2 cells.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

G. Raising Monoclonal Antibodies Specific for Intimin

Monoclonal antibodies directed against intimin are used to passivelyprotect a patient against colonization by EHEC (or bacteria expressingintimin-like proteins). Monoclonal antibodies are generated from mousecells, and the specificity of these antibodies are changed for use inhumans. G. Winter et al., “Man-made antibodies,” Nature, 349: 293-299(1991); P. T. Jones et al., “Replacing the complementarity-determiningregions in a human antibody with those from a mouse,” Nature, 321:522-525 (1986); P. Carter et al., “Humanization of an anti-p185^(HER2)antibody for human cancer therapy,” Proc. Natl. Acad. Sci. USA, 89:4285-4289(1992). Monoclonal Abs represent a more “pure” antibody foradministration to a patient.

1. Generation of Anti-Eae Monoclonal Antibodies

Two examples of methods for generating anti-intimin monoclonalantibodies are described below.

a. Method 1

Generation of Anti-Eae mAbs

The procedure outlined in Harlow, E. and D. Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor, N.Y. (1988) is followed withmodifications. Nine week old female BALB/c (Harlan Spraque-Dawley,Indianapolis, Ind.) are used for the production of monoclonalantibodies. Prior to immunization, a serum sample is obtained from eachmouse via the retro-orbital sinus. The whole blood is placed into amicrofuge tube and allowed to cool at 4° C. for between 4 and 16 hours.Serum is prepared by centrifugation of the whole blood at 1000-1200×gfor 15 minutes at 10-15° C. The serum is transferred to new microfugetubes using a micropipettor and sterile pipets tips. The serum is storedat −20° C. until use.

The antigen is obtained from SDS-PAGE gels of RIHisEae, obtained asdescribed above in Example II. The high molecular weight intimin band isexcised with a razor, as described above in Example IX, section A, part4. One mg of RIHisEae is run onto four MiniProtean II gels (BioRad,Richmond, Calif.) for this purpose. Protein excised from the gels aremade into a slurry in approximately 8 mls of phosphate buffered saline(PBS) using a mortar and pestle. On experimental day 0, a 0.8 ml portionof the slurry is mixed with 1.2 mls of complete Freund's adjuvant (CFA)and injected in 0.2 ml aliquots subcutaneously into each of four mice. A0.5 ml portion of the slurry is mixed with 0.5 mls of RIBI T-700adjuvant (RIBI Immunochem, Hamilton, Mo.) and 0.2 mls is injected intoeach of four additional mice.

Mice receive booster injections on experimental days 21 and 42. Theantigen is prepared as described above, with the exception thatincomplete Freund's adjuvant (IFA) is used instead of CFA.

Serum samples are obtained as described above on experimental days 14,35 and 49.

Serum samples are tested by immunoassay (as described below) to identifymice producing serum with the strongest response to Eae, as would berecognized to those skilled in the art. The reactivity of the serumsamples is verified by Western blot analysis as described above inExample IX, section A, part 4. Three days prior to fusion (onexperimental day 59), the mouse chosen for fusion is immunized with a50% mixture of supernatant from the intimin slurry in PBS. A total of0.1 mls of this slurry is injected intravenously via the tail vein.

Spleen cells from the chosen mouse are fused with SP2/0 myeloma cells(Cat # CRL1581 American Type Culture Collection, Rockville, Md. 20850,301-881-2600). A ratio of 10 spleen cells: 1 myeloma is used for thefusion. Fusion is accomplished by the use of polyethylene glycol (Cat#783 641 Boehringer-Mannheim Corp., 9115 Hague Road, PO Box 50414,Indianapolis, Ind. 46250, 800-262-1640). Fused cells are distributedinto 96-well tissue culture dishes for growth. Hybridomas are selectedby growth of the cultures for 10 days in medium containing hypoxanthine,aminopterin and thymidine. Hybridomas secreting anti-intimin specificantibodies are identified from the 96-well tissue culture dishes byimmunoassay as described below. Cultures positive for antibodiesreactive with Eae are expanded by transfer to 24-well dishes, retestedfor reactivity with Eae by immunoassay and cloned twice by limitingdilution.

Immunoassay (ELISA) of Mouse Polyclonal Anti-Intimin Serum and HybridomaSupernatants (Anti-Intimin Monoclonal Antibodies)

A three ml portion of the intimin slurry used for immunization iscentrifuged at approximately 1000×g for 15 minutes at room temperature.A sample of the resulting, clarified supernatant is used to coatimmunoassay plates. Briefly, the intimin-containing supernatant isdiluted 1:300 in PBS and used as a coating antigen. Nunc MAXISORP™Stripwells, which provide a charged polystyrene ELISA surface with highaffinity for polar or hydrophilic groups (Nunc, Naperville, III.), arecoated with 100 μl/well of the diluted supernatant for 2-24 hours atroom temperature.

Unbound material is washed from the wells with four washes of PBScontaining 0.5% TWEEN® 20(ICI Americas, Inc., Wilmington, Del.),(PBS-T). For assays of serum samples, multiple dilutions of each sampleare prepared in PBS-T and added to replicate wells. For assays ofculture supernatants from 96-well dish cultures, each supernatant isdiluted 1:2 in PBS-T and added to a single well. Supernatants from24-well dish cultures are also diluted 1:2 in PBS-T and tested induplicate. Assays of serum samples include a buffer control and a knownpolyclonal anti-intimin control. Assays of supernatants include a buffercontrol, medium control and a known polyclonal anti-intimin control.

Serum and supernatants are allowed to incubate in a draft-freeenvironment at room temperature for 30-60 minutes on the intimin-coatedwells and unbound antibodies and extraneous material (such as serumproteins) are washed from the wells with four washes of PBS-T. Each wellthen receives 100 μl of rabbit anti-mouse IgG (gamma specific)-HRP(Zymed, South San Francisco, Calif.), diluted 1:4000 in PBS-T.

The plates are again allowed to incubate in a draft-free environment atroom temperature for 30-60 minutes. Each well then receives 100 μl ofone-component TMB substrate solution (teramethylbenzidine) (Kirkegaardand Perry Labs, Gaithersburg, Md. 20878, 301-948-7755). The reaction isallowed to proceed for 15 minutes in the dark and then stopped by theaddition of 80 μl/well of TMB stop reagent (Kirkegaard and Perry Labs,Gaithersburg, Md. 20878, 301-948-7755).

b. Method 2

The procedure outlined in Harlow, E. and D. Lane, Antibodies. ALaboratory Manual. Cold Spring Harbor, N.Y. (1988) is followed: Five 4-to 5-week old female BALB/cJ mice are prebled, and immunizedintraperitoneally with 25 μg RIHisEae suspended in 100 μl of TITERMAX®.Mice are boosted twice in two week intervals, intraperitoneally with 25μg RIHisEae suspended in 100 μl of TITERMAX®. Seven days after eachboost, blood (˜300-500 μl) is collected from the tail vein. Sera areassayed for the presence of anti-RIHisEae antibody by ELISA (asdescribed above).

Mice producing high titers of anti-RIHisEae antibodies are boosted bothintravenously and intraperitoneally with 25 μg of RIHisEae in 100 μl ofPBS, sacrificed three days later, and sera collected. Spleen cells areisolated and fused to Sp2/0-Ag mouse myeloma cells (ATCC #CRL1581) at aratio of 10 spleen cells to 1 myeloma cell. Fused cells are distributedinto microdilution plates, and culture supernatants are assayed by ELISAafter 34 weeks of culture for RIHisEae antibodies. Cultures positive forproduction of anti-RIHisEae antibodies are expanded and cloned twice bylimiting dilution.

2. Determination of Whether anti-RIHIsEae mAbs Recognize Conformationalor Linear Epitopes

Reactivities of the mAbs are compared by: 1) ELISA in which nativeRIHisEae is used as the adsorbent; and 2) immunoblot of RIHisEaedenatured and separated by SDS-PAGE. Several pools of mAbs areobtained: 1) those that recognize only conformational epitopes and reactpositively by ELISA but not by immunoblot analysis; 2) those thatrecognize linear epitopes and react in both assays; and 3) those thatrecognize linear epitopes and react positively by immunoblot analysis,but not by ELISA. In addition, colony immunoblots of unlysed cells aredone to determine if the mAbs recognize Eae expressed on the surface ofthe wild type strain 86-24.

3. Testing of Anti-Eae mAbs for Capacity to Block Adherence of Strain86-24 to HEp-2 Cells

Strain 86-24 is subjected to a qualitative adherence assay on HEp-2cells and tested in parallel with bacteria that have been pre-incubatedwith various dilutions of anti-RIHisEae mAbs.

Selected adherence-blocking and conformational mAbs are subjected toisotype determination (IMMUNOPURE® mAb Typing Kit, and isotyping ELISAkit, Pierce, Rockford, Ill.). Unique antibodies are then purified byaffinity chromatography on a Protein G Sepharose column (Pharmacia,Piscataway, N.J.). The resulting affinity-purified mAbs are re-testedfor capacity to block adherence of strain 86-24 to Hep-2 cells to ensurethat the antibody remains functional after purification.

H. Use of Polyclonal and Monoclonal Anti-intimin Antibodies inDiagnostic kits.

Diagnostic kits can be used to detect intimin-expressing bacteria,preferably EHEC. A general description of the preparation and use ofsuch kits is provided in copending U.S. Application Ser. No. 08/412,231,filed Mar. 10, 1995, the disclosure of which is incorporated herein byreference.

EXAMPLE VIII

High titer polyclonal anti-intimin antisera are elicited uponintraperitoneal injection of 25 μg RIHisEae into mice and rabbits, usingTITERMAX® adjuvant (CytRx Corp., 154 Technology Parkway, TechnologyPark/Atlanta, Norcross, Ga. 30092, 800-345-2987). Testing of antibodytiter and an antibody effectiveness assay are shown. Monoclonalantibodies are also described.

A. Making Polyclonal Antibodies

The technique of Harlow, E. and D. Lane (eds) Antibodies—a LaboratoryManual.Cold Spring Harbor, N.Y. (1988) may be followed. The generalprocedure is outlined herein. Take pre-bleeds of each mouse to beimmunized: Bleed from the tail vein into an eppendorf tube. Incubate at37° C. for 30 min, stir gently with a sterile toothpick (to loosen theclot), store overnight at 4° C. In the morning, spin 10 min/10,000 rpmin the microfuge, and collect the serum (i.e., supernatant; red bloodcells are the pellet). Store the serum at −20° C. The sera obtained willbe used as a negative control after the mice are immunized.

Inject a BALB/c mouse intraperitoneally with 25 μg of RIHisEae (usingTITERMAX® adjuvant, according to the instructions of the manufacturer(CytRyx Corp., 154 Technology Pkwy., Norcross, GA. 30092, 800-345-2987).Wait 2 weeks, boost with an identical shot, wait 7 days and bleed fromthe tail vein into an eppendorf tube. Incubate at 37° C. for 30 min,stir gently with a sterile toothpick (to loosen the clot), storeovernight at 4° C. In the morning, spin 10 min/10,000 rpm in themicrofuge, and collect the serum. Store the sera at −20° C.

B. ELISA to Test Titer of Abs

The technique of Harlow, E. and D. Lane (eds) Antibodies—a LaboratoryManual. Cold Spring Harbor, N.Y. (1988) may be followed. The generalprocedure is outlined below:

(1) bind. RIHisEae to plastic microtiter plates at 50 ng/well in PBS.Incubate 2 h/RT (room temp) or overnight at 4° C.

(2) wash plate 2×with PBS.

(3) block wells with 100 μl blocking solution [3% bovine serum albumin(Sigma Chemical, St. Louis, Mo.), 0.02% sodium azide (Sigma) inPBS—store stock at 4° C.] for 1-2 h at RT.

(4) wash plate 2×with PBS.

(5) primary Ab=50 μl test sera diluted in blocking solution for example,start with 1:50 and do eleven 1:2 dilutions, or start with 1:50 and doeleven 1:10 dilutions), incubate 2 h/RT.

(6) wash 4×with PBS.

(7) secondary Ab=goat horseradish-conjugated anti-mouse Ig, affinitypurified (Boehringer Mannheim Corp., 9115 Hague Rd., P.O. Box 50414,Indianapolis, Ind. 46250, 800-262-1640). Add secondary Ab diluted 1:500in blocking solution without azide. Incubate 1 h/RT.

(8) wash 4×with PBS.

(9) add 100 μl TMB Peroxidase substrate to each well (prepared accordingto the instructions of the manufacturer, BioRad Labs, 3300 RegattaBlvd., Richmond, Calif. 94804). Allow blue color to develop (no morethan 10 min). Stop the reaction with 100 μl H₂SO₄. Read the plate at 450nm.

A titer is defined as an absorbance value ≧0.2 units above that obtainedfor mouse pre-immune sera.

Anti-intimin Abs obtained from animals may be used clinically if onechanges the specificity of the antibody to human. Such techniques arewell known to those of ordinary skill in the art. G. Winter et al.,“Man-made antibodies,” Nature, 349: 293-299 (1991); P. T. Jones et al.,“Replacing the complementarity-determining regions in a human antibodywith those from a mouse,” Nature, 321: 522-525 (1986); P. Carter et al.,“Humanization of an anti-p185^(HER2) antibody for human cancer therapy,”Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992). Such antibodies may begiven to the sibling of an infected patient to reduce the risk ofinfection of the sibling.

C. Assay for Blocking of Bacterial Binding by Antibodies to Intimin

To assess the effect of anti-intimin antibodies on EHEC adherence, mouseor rabbit anti-intimin antisera (or normal sera as controls) are addedto EHEC bacteria suspended in adherence media, and the bacteria-antiseramixtures are incubated at 37° C. for thirty minutes prior to infectionof HEp-2 cells. Antisera are maintained in the adherence mediathroughout the assay. Adherence and related sequelae are microscopicallyobserved using GIEMSA and FITC-phalloidin (FAS) staining as describedabove.

To assess the effect of anti-intimin antibodies on adherence of otherbacteria having intimin-like proteins, mouse or rabbit anti-intiminantisera (or normal sera as controls) are added to EHEC bacteriasuspended in adherence media, and the bacteria-antisera mixtures areincubated at 37° C. for thirty minutes prior to infection of HEp-2cells.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

D. Raising Monoclonal Antibodies to Intimin

Monoclonal antibodies directed against intimin are used to passivelyprotect a patient against colonization by EHEC (or bacteria expressingintimin-like proteins). Monoclonal antibodies are generated from mousecells, and the specificity of these antibodies are changed for use inhumans. G. Winter et al., “Man-made antibodies,” Nature, 349: 293-299(1991); P. T. Jones et al., “Replacing the complementarity-determiningregions in a human antibody with those from a mouse,” Nature, 321:522-525 (1986); P. Carter et al., “Humanization of an anti-p185^(HER2)antibody for human cancer therapy,” Proc. Natl. Acad. Sci. USA, 89:42854289(1992). Monoclonal Abs represent a more “pure” antibody foradministration to a patient.

1. Generation of anti-Eae mAbs: The procedure outlined in Harlow, E. andD. Lane, Antibodies. A Laboratory Manual. Cold Spring Harbor, N.Y.(1988) is followed. Five 4- to 5-week old female BALB/cJ mice areprebled, and immunized intraperitoneally with 25 μg RIHisEae suspendedin 100 μl of TITERMAX®. Mice are boosted twice in two week intervals,intraperitoneally with 25 μg RIHisEae suspended in 100 μl of TITERMAX®.Seven days after each boost, blood (˜300-500 μl) is collected from thetail vein. Sera are assayed for the presence of anti-RIHisEae antibodyby ELISA (as described above).

Mice producing high titers of anti-RIHisEae antibodies are boosted bothintravenously and intraperitoneally with 25 μg of RIHisEae in 100 μl ofPBS, sacrificed three days later, and sera collected. Spleen cells areisolated and fused to Sp2/0-Ag mouse myeloma cells (ATCC #CRL1581) at aratio of 10 spleen cells to 1 myeloma cell. Fused cells are distributedinto microdilution plates, and culture supernatants are assayed by ELISAafter 3-4 weeks of culture for RIHisEae antibodies. Cultures positivefor production of anti-RIHisEae antibodies are expanded and cloned twiceby limiting dilution.

2. Determination of whether anti-RIHisEae mAbs recognize conformationalor linear epitopes: Reactivities of the mAbs are compared by: 1) ELISAin which native RIHisEae is used as the adsorbent; and 2) immunoblot ofRIHisEae denatured and separated by SDS-PAGE. Several pools of mAbs areobtained: 1) those that recognize only conformational epitopes and reactpositively by ELISA but not by immunoblot analysis; 2) those thatrecognize linear epitopes and react in both assays; and 3) those thatrecognize linear epitopes and react positively by immunoblot analysis,but not by ELISA. In addition, colony immunoblots of unlysed cells aredone to determine if the mAbs recognize Eae expressed on the surface ofthe wild type strain 86-24.

Testing of anti-Eae mAbs for capacity to block adherence of strain 86-24to HEp-2 cells: Strain 86-24 is subjected to a qualitative adherenceassay on HEp-2 cells and tested in parallel with bacteria that have beenpre-incubated with various dilutions of anti-RIHisEae mAbs.

Selected adherence-blocking and conformational mAbs are subjected toisotype determination (IMMUNOPURE® mAb Typing Kit, Pierce, Rockford,Ill.). Unique antibodies are then purified by affinity chromatography ona Protein G Sepharose column (Pharmacia, Piscataway, N.J.). Theresulting affinity-purified mAbs are re-tested for capacity to blockadherence of strain 86-24 to Hep-2 cells to ensure that the antibodyremains functional after purification.

EXAMPLE X

Agrobacterium Tumefaciens—mediated Transformation of Various Plants forExpression of Intimin.

Plants are transformed to express intimin, or functional portions ofintimin, and fed to patients. As those of ordinary skill in this artwould recognize, the intimin may be his-tagged as described in ExampleI, or not. In addition, the intimin may be a fusion protein comprisingintimin and one or more antigens against which an immune response isdesired to be elicited. (See Example VII.)

Any plant tissue may be transformed with a vector of the presentinvention. The term “organogenesis,” as used herein, means a process bywhich shoots and roots are developed sequentially from meristematiccenters; the term “somatic embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially). Exemplary tissue targets include leaf disks,pollen, embryos, somatic embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristems, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

A. Construction of the Plasmid Containing the eae Gene

The present example uses the vector, pKYLX 71S² (FIG. 17). This vectoris obtained from David Hunt, Dept. of Crop Science, University ofKentucky, Lexington, Kentucky but those in the art will recognize thatother available vectors may be used or constructed. This vector is anAgrobacterium tumefaciens binary vector containing kanamycin in vivoselectable marker gene (NPTII). A binary vector system contains twoplasmids. A tumor-inducing (Ti) plasmid contains DNA (t-DNA), into whichthe desired coding region is inserted in a multiple cloning region. Theother plasmid contains vir genes, which are virulence genes enabling thet-DNA to enter plant cells and integrate into the genome. The pKYLX 71S²vector places the desired coding sequence under the control of adoubled-enhanced cauliflower mosaic virus 35S (CaMV 35S or simply 35S)promoter. In this case, the doubled-enhanced promoter is two ribosomalpromoters in tandem.

Agrobacterium vectors are useful for introducing foreign genes into avariety of plant species and particularly useful for the transformationof dicots. Numerous Agrobacterium vectors are known. See, e.g., U.S.Pat. No. 4,536,475 to Anderson, U.S. Pat. No. 4,693,977 to Schliperoortet al.; U.S. Pat. No. 4,886,937 to Sederoff et al.; T. Hall et al., EPOApplication 0122791; R. Fraley et al., Proc. Natl. Acad. Sci. USA 84,4803 (1983); L. Herrera-Estrella et al., EMBO J. 2, 987 (1983); G.Helmer et al., Bio/Technology 2, 5201 (1984); N. Murai et al., Science222, 476 (1983).

The his-eae gene (obtained as described above in Example 1) is ligatedinto vector pKYLX 71S², creating the plant transformation vector pINT(FIG. 17), using recombinant techniques well known to those ordinarilyskilled in the art.

The his-eae gene (obtained as described above in Example I) is ligatedinto vector pKYLX 71S², creating the DNA construct pINT (FIG. 17). Suchvectors containing heterologous DNA can be constructed using recombinanttechniques well known to those ordinarily skilled in the art. Forexample, DNA from pEB313 (described above in Example I) is prepared withthe use of a QIAGEN DNA extraction kit (QIAGEN, Chatsworth, Calif.). Thehis-eae gene is isolated by digestion of pEB313 with XhoI and NheI,separation on an agarose gel, followed by excision of the 3147 bpeae-containing band with a razor. The purified DNA is extracted from theagarose with GENECLEAN (Bio101, LaJolla) and ligated into pKYLX71s²digested with XhoI and XbaI. Liagated plasmids are transformed intoDH5αF'Tn5/ac/_(Q), and transformants verified for the presence ofinserted DNA by digestion with appropriate restriction enzymes. See alsoExample I. Any publicly available Agrobacterium tumefaciens strains maybe used, but strains LBA4404, GV3850 or EHA105 (obtainable from StanleyGelvin, Purdue University, West Lafayette, Ind.) are preferred. The pINTplasmid is transferred to A. tumefaciens using calcium-chloride ions,followed by freeze-thaw transformation, electroporation, or othermethods well known to the art. See, for example, Hanahan, D., J. Mol.Biol. 166:577-80 (1983).

B. Transformation of Tobacco.

Tobacco is used very commonly as a model for plant transformation. Ageneral assumption that had been confirmed by many empirical studies isthat if a transgene is expressed in tobacco then it will be expressed inanother dicot plant. Recognizing that tobacco is not an edible plant, ifa recombinant intimin is produced in tobacco, then it will beexpressible in an edible plant such as canola.

Tobacco (Nicotiana tabacum) cultivar ‘Xanthi’ is transformed byAgrobacterium-mediated transformation using a standard and efficientinfection protocol (Schardl et al., Gene 61: 1-11 (1987)). Briefly,tobacco leaf discs 0.5 cm in diameter are wounded and exposed to theAgrobacterium tumefaciens containing pINT. Plants are regenerated usingan organogenic method under kanamycin selection (200 mg/L) in tissueculture.

Two hundred 0.5 cm leaf disks of tobacco are exposed to Agrobacteriumtumefaciens harboring pINT. Tissue culture and plant regenerationconditions follow Schardl. et al 1987. Shoots are formed directly fromwounded leaf disks under 200 mg/L kanamycin selection and 400 mg/LTimentin to kill Agrobacteria. This system is both highly efficient andnot leaky (non-transgenic “escapes” are extremely rare). Three hundredand seventy-five shoots are produced from the experiment and 120 ofthese are arbitrarily selected for rooting on hormone-free media. Allplants are morphologically normal and fertile. These results fall withinthe typical transformation efficiencies using this system. The hightransformation frequencies and the fact that the plants are healthyindicate that intimin is not toxic, and does not interfere with normalplant development and function.

To determine that his-eae is stably integrated into the plant, leaftissue is processed according to well known methods for Southern (DNA)blot analysis (Stewart et al., Plant Physiol. 112:121-129 (1996);Stewart, C. N., Jr. and Via, L. E., Biotechniques 13: 748-751 (1993). etal.), PCR analysis (Stewart et al., Plant Physiol. 112:121-129 (1996),and Western (protein) blot analysis (Stewart et al., Plant Physiol.112:121-129 (1996). Southern blotting is performed to verify thepresence of the eae gene in the DNA of the leaf tissue. PCR analysis isperformed to verify the presence of the eae gene, as well. Primers usedmay include those mentioned above in Example I part D. For example, PCRamplification of putative eae-containing plant DNA using the primers MW1(=5′GTACGGATCCGAATTCATTTGCAAATGGTG 3′) SEQ ID NO:6 and MW2(=5′GTACGGTACCTGATCAATGAAGACGTTATAG 3′) SEQ ID NO:7 results in anexpected 734 bp band when resolved on an agarose gel. Western blotanalysis is performed to determine levels of expression of his-intiminwithin the leaf. Once expression of his-intimin exceeds 0.1% of totalplant protein, the his-intimin protein is isolated using a one stepnickel column purification (Stolz et al., Plant Journal 6: 225-233(1994)). The purification method of Example II is an alternative.His-intimin is subjected to the adherence assay shown in Example III toverify functional binding.

Preparation of Extracts

Protein extracts are recovered from putative transgenic tobacco plantsas follows: Five hundred microliters of extraction buffer (20 mls 0.5 MTris HCl pH 8.0, 4 mls 0.5 M EDTA pH 8.0, 36 mls glycerol, 20 mlsβ-mercaptoethanol) or 500 μl of phosphate buffer (50 mM Na-phosphate pH7.8, 300 mM NaCl) are added to 0.2 g of leaf tissue, the mixture ishomogenized on ice, and then clarified by a pulse spun in a microfuge.The supernatant recovered, placed into a fresh eppendorf tube and storedat −70° C. Protein extracts are tested by Western blot analysis asdescribed in Stewart et al., Plant Physiol. 112:121-129 (1996), usingany of the monoclonal or polyclonal anti-intimin antibodies described inExample IX above.

If expression of his-intimin is hampered by the high Adenine (A)/Thymine(T) content of eae, the gene is rebuilt using methods outlined in Adanget al. (Adang et al., 1993), incorporated herein by reference. Briefly,for the segment of eae contained within pINT, the nucleotide sequence isanalyzed to determine the codon specifying each amino acid in theintimin fragment. Capitalizing on the redundancy of the genetic code,the codons are rewritten to replace A or T with C or G without changingthe amino acid specified by the codon; thus, codons of heterologous DNA,of bacterial origin, for example, are replaced with codons that arepreferred for expression in a plant cell. The preferred substitutionsare chosen according to the codon preference for the plant species beingused. For instance, see Murray, E. E., et al., Nucleic Acids Res. 17(2): 477498 (1989); Dinesh-Kumar, S. P. and Miller, W. A., Plant Cell 5(6): 679-692 (1993); Kumar, P. A. and Sharma, R. P., J. Plant Chemistryand Plant Biotechnol. 4 (2): 113-115 (1995).

The synthetic gene sequence is divided into regions of 200-300 bp andoligonucleotides are synthesized for each region. Oligonucleotides aresynthesized by Research Genetics, Huntsville, Ala. The ordinarilyskilled artisan will recognize that the oligonucleotides may besynthesized in many other commercial laboratories. The oligonucleotidesare ligated according to the method set forth in Adang et al. Plant Mol.Biol. 21: 1131-1145 (1993) and the reconstructed gene is inserted intopKYLX 71S². The resulting plasmid, pINTrecCA, is used to transform anyone of the preferred Agrobacterium tumefaciens strains LBA4404, GV3850or EHA105. The tobacco is transformed and the expression productanalyzed as set forth above.

C. Transformation of Other Plants

Tobacco is not edible, therefore any intimin expressed in the tobaccoplant must be purified to remove alkaloids for safe use. Plants that arepart of the normal diet for the patient and are easily engineered arepreferred. As examples, which are not intended to be limiting, forcattle, swine, and mutton, either canola (Stewart et al., Insect controland dosage effects in transgenic canola, Brassica napus L.(Brassicaceae), containing a synthetic Bacillus thuringiensis Cryla(c)gene (Stewart et al., Plant Physiol. 112:115-120 (1996)) or alfalfa(Thomas et al., Plant Cell Rep. 14: 31-36 (1994)) may be transformed toexpress intimin or an intimin fusion protein. For humans, canola orcarrot (Droge et al., 1992) may be transformed.

The patient should ingest the intimin-containing plant or theintimin-containing portion of a plant. The injested portion may be aplant leaf, root, fruit, berry, seed, tuber, corn, inflorescence, stemetc. or combination thereof. The injested portion may also be extractedfrom a plant as a plant derivative such as, for example, flour, meal,slurry, infusion, paste, juice, powder, cake, or pellet. The injestedportion may be further incorporated into a complex feedstock orfoodstuffs according to techniques commonly known in the art.

Haq et al., Science, 268:714-716, 1995 have made transgenic tobacco andpotato plants using genes encoding the B subunit of the ETEC heat-labileenterotoxin (LT-B) or this gene with a microsomal retention sequence(SEKDEL (SEQ ID NO:27)). Both plants expressed the fusion proteins,which formed oligomers that bound the natural ligand. When mice weretube fed a crude soluble extract of tobacco leaves expressingLT-B-SEKDEL, they produced serum and gut mucosal anti-LT-B immunoglobinsthat neutralized the enterotoxin in cell protection assays. Mice fedpotato tubers expressing LT-B-SEKDEL also developed serum and gutmucosal immunity specific for LT-B.

Plants which may be employed in practicing the present invention include(but are not limited to) tobacco (Nicotiana tabacum), potato (Solanumtuberosum), soybean (glycine max), sunflower, peanuts (Arachishypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus),cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocosnucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), corn (Zea mays),wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers.Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Pisum spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azelea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (dianthus caryophyllus), poinsettia(Euphorbia pulcherima), and chrysanthemum. Gymnosperms which may beemployed to carrying out the present invention include conifers,including pines such as loblollypine (Pinus taeda), Douglas-fir(Pseudotsuga menziesil); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); and redwood (Sequoia sempervirens).

EXAMPLE XI

Gene Gun-mediated Transformation of Various Plants, for Example,Monocots Like Corn, Wheat, and Rice

Another method of transforming plants to express intimin or intiminfusion proteins is provided. The plasmid described in Example IX, pINT,is coated onto microprojectiles (microparticles). Specifically, 1 μg ofpINT is coated onto 10 mg of gold microparticles 1 micron in diameter(Biorad Laboratories tungsten particles obtained from Sylvania may alsobe used). Next, 5 μl of 10⁸ cells of Agrobacterium tumefaciens areovercoated onto the pINT coated microprojectile. One mg of thedoublecoated microprojectiles is loaded into the PDS 1000-He gun,according to manufacturer. (Biorad Laboratories) One gram of soybeanembryos initiated from immature cotyledons is bombarded with thedoublecoated microprojectiles at 1000 psi. Bombarded embryos are grownunder selection for kanamycin (200 mg/L) in tissue culture. They areallowed to mature and germinate and are fed to animals, such as pigs.

The same method may be used to transform bananas. The above method mayalso be accomplished without the step of adding Agrobacteriumtumefaciens. Reconstruction of the eae gene or desired gene region maybe accomplished as set forth in Example IX.

EXAMPLE XII

Expression of Intimin as Chimeric Virus Particles (CVP) Fusion Proteins

Another method of transforming plants to express intimin is through theuse of a recombinant plant virus. This method is preferred for the rapiddevelopment and delivery of inexpensive oral vaccines. An intimin orintimin-like protein, or portion thereof, or recombinant fusion proteinthereof, may be expressed using a plant recombinant viral infection andexpression system such as that described by Dalsgaard and coworkers(Dalsgaard et al., Nature Biotechnology, 15:248-252 (1997), thedisclosure of which is incorporated herein by reference), and as furtherdescribed by Arntzen (Nature Biotechnology, 15:221-222 (1997), thedisclosure of which is incorporated herein by reference). In a preferredembodiment, DNA encoding the intimin or intimin-like protein, or portionthereof, or recombinant fusion protein thereof, is recombinatorilyinserted into a gene encoding a coat protein thereof, and optimally isexpressed as a fusion protein with the coat protein. Preferably, theintimin or intimin-like protein, or portion thereof, or fusion proteinthereof, is antigenic. The resulting recombinant viral genome is thenpropagated in a plant, portion or a plant, plant cell or plant extract,resulting in the expression of intimin or intimin-like protein, orportion thereof, or recombinant fusion protein. The intimin orintimin-like protein, portion thereof, or recombinant fusion proteinthus produced by a viral vector may be ingested, extracted, enriched,purified or otherwise applied to the methods disclosed elsewhere herein.

EXAMPLE XIII

Expression of His-intimin in Another Bacterial Species that does notNormally Express it

The intimin or intimin-like protein, portion thereof, or recombinantintimin fusion protein may be expressed in a host bacterium and the hostbacterium may be given as a vaccine. For example, Su. G-F. et al.Microbial Pathogenesis 13:465 (1992), the materials and methods segmentfor which are hereby incorporated by reference, show the Shiga toxin Bsubunit fused to the 23 kDa C-terminus of E. coli hemolysin A (HIyA)which was then used to export it from an attenuated carrier strain ofSalmonella typhimurium aroA (SL3261). The expression of this gene fusionwas under constitutive control or under the control of an iron-regulatedpromoter. Oral and intraperitoneal immunization of mice with the hybridstrain resulted in significant B-subunit specific mucosal and serum Abresponses. Both cytoplasmic expression and extracellular export from theantigen carrier strain were shown to result in antigen-specific immuneresponses.

In addition, Pozzi, G. et al. Infect. Immun. 60:1902 (1992), thedisclosure of which is hereby incorporated by reference, show a systemin which a foreign antigen is fused to the C-terminal attachment motifof the fibrillar M protein from Streptococcus pyogenes. The fusionprotein is expressed on the surface of S. gordonii, a commensal organismof the oral cavity. In this study, the E7 protein of humanpapillomavirus type 16 was chosen as the foreign antigen and was fusedto the fibrillar M protein. Mice were infected subcutaneously. The M-E7fusion protein expressed on the surface of S. gordonii was shown to beimmunogenic for both aspects of the fusion protein in. mice asdemonstrated by Western blot analysis using sera pooled from infectedmice.

In addition, Schafer, R. et al. J. Immunol. 149:53 (1992), the materialsand methods segment for which are hereby incorporated by reference,showed Listeria monocytogenes expressing E. coli β-galactosidase(foreign antigen) was used as a live vaccine vector. BALB/c mice wereimmunized orally or intraperitoneally. Spleen cells harvested one weekafter oral or five weeks after oral or peritoneal infection (boosted at4 weeks) showed β-galactosidase-specific cytotoxic T lymphocyteresponses. Individual serum samples from mice immunizedintraperitoneally or intravenously were tested for anti-β-galactosidaseantibodies; approximately 11% had positive titers for these antibodies.These results show that both oral and parenteral immunization with thisspecies results in a cellular immune response to a foreign protein.

An example of practicing the present invention according to the methodof Su et al. is shown herein. In this example, eae (or his-eae) isplaced under the control of the aerobactin promoter and fused to a DNAfragment encoding the 23 C-terminal hemolysin A (HIyA) signal domain,which targets the desired protein extracellularly. The aerobactinpromoter is activated under iron limitation, a condition found in theintestinal mucosal environment, which promotes gene expression in vivo.The plasmid used to promote such expression is a medium copypBR322-based plasmid, which confers a greater degree of immunity than ahigher copy plasmid, such as pUC18. The bacterial host for expression ofthe foreign protein is Salmonella typhimurium aroA strain SL3261 pLG575,an attenuated derivative of an intestinal pathogen (available fromKenneth Timmis, Dept. of Microbiology, GBF-National Research Centre forBiotechnology, Braunschweig D-3300, Germany, as described in Su et al.).This strain also contains plasmid pLG575, encoding the hlyB and hlyDgenes which are required for export of the intimin-HIyA protein.

Briefly, (FIG. 18) the BamHI StxB fragment is dropped out of the plasmidpSU204 (available from Kenneth Timmis, Dept. of Microbiology,GBF-National Research Centre for Biotechnology, Braunschweig D-3300,Germany, the above reference), creating pSU2004. A BamHI DNA fragmentencoding intimin or an intimin fusion protein is constructed by PCR suchthat the coding region is in the correct reading frame with both theaerobactin promoter contained on pSU2004, as well as the hlyA codingregion. The BamHI eae fragment is ligated into pSU2004, resulting inpAero-Eae. This plasmid is transformed into the restriction-negativemodification-positive S. typhimunum strain SL5283 (available fromKenneth Timmis) according to the method Hanahan, D. J., Mol. Biol.166:577-580 (1983). Such a step modifies the pAero-Eae DNA bymethylation, and plasmid DNA purified from this strain transformsS.typhimurium aroA strain SL3261 pLG575 more efficiently. MethylatedpAero-Eae DNA is transformed into S. typhimurium aroA strain SL3261pLG575 by the method of Hanahan, D. J., Mol. Biol. 166:577-580 (1983).

Eight to ten week old female BALB/c mice are fed the immunogenicbacterial host as follows. Bacteria are grown overnight at 37° C. in LBbroth containing 100 μg/ml ampicillin, inoculated at a dilution of 1:50into fresh media, and grown until the OD₆₀₀=0.3. The promoter is theninduced with 2,2′-bipyridyl (100 μM final concentration) for 3 hours.The culture is washed and resuspended in PBS for feeding.

Two doses of bacteria are fed 4 days apart with 10¹⁰ CFU, with the aidof a feeding tube. Twenty one days after the first feeding, a boosterinjection is given. Mice are sacrificed one week later and show amucosal and humoral immune response.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

We claim:
 1. A plant cell expressing intimin, comprising a plant celltransformed with a plant transformation vector comprising heterologousDNA encoding intimin, under the control of a plant promoter, such thatthe intimin which is expressed from the heterologous DNA retains bindingfunction, and wherein the heterologous DNA further encodes a histidnetag.
 2. A method for producing a plant which expresses intimin,comprising: a) transforming a plant cell with a plant transformationvector comprising heterologous DNA, encoding intimin, under the controlof a plant promoter, wherein the intimin retains binding function, andwherein the heterologous DNA, further encodes a histidine tag; b)regenerating a plant from said transformed plant cell, wherein theregenerated plant expresses the intimin; and c) utilizing theregenerated plant expressing intimin as a source of intimin.
 3. A methodof claim 2, further comprising enriching or purifying the intimin fromthe regenerated plant.
 4. A plant cell of claim 1, wherein the plantcell resides in a plant tissue capable of regeneration.
 5. A plant cellof claim 1, wherein the plant transformation vector is an Agrobacteriumvector.
 6. A plant cell of claim 1, wherein the plant transformationvector is a viral vector.
 7. A method of claim 2, wherein the planttransformation vector is an Agrobacterium vector.
 8. A method of claim2, wherein the plant transformation vector is a microparticle.
 9. Amethod of claim 8, wherein the plant cell is transformed by bombardmentwith the microparticle.
 10. A method of claim 2, wherein the planttransformation vector is a viral vector.
 11. A method of claim 2,wherein the regenerated plant comprises shoots.
 12. A method of claim 2,wherein the regenerated plant comprises roots.
 13. A method of claim 3,wherein the enriching or purifying of the intimin comprises using atleast one of high performance liquid chromatography (HPLC), gel columnchromatography, and SDS-PAGE.